Dosages of Immunoconjugates of Antibodies and SN-38 for Improved Efficacy and Decreased Toxicity

ABSTRACT

The present invention relates to therapeutic immunoconjugates comprising SN-38 attached to an antibody or antigen-binding antibody fragment. The antibody may bind to EGP-1 (TROP-2), CEACAM5, CEACAM6, CD74, CD19, CD20, CD22, CSAp, HLA-DR, AFP or MUC5ac and the immunoconjugate may be administered at a dosage of between 4 mg/kg and 24 mg/kg, preferably 4, 6, 8, 9, 10, 12, 16 or 18 mg/kg. When administered at specified dosages and schedules, the immunoconjugate can reduce solid tumors in size, reduce or eliminate metastases and is effective to treat cancers resistant to standard therapies, such as radiation therapy, chemotherapy or immunotherapy.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/204,698, filed Mar. 11, 2014, which was a divisional of U.S.application Ser. No. 13/948,732 (now U.S. Pat. No. 9,028,833), filedJul. 23, 2013, which claimed the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Applications 61/736,684, filed Dec. 13, 2012, and61/749,548, filed Jan. 7, 2013.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 17, 2013, isnamed IMM340US1_SL.txt and is 60,438 bytes in size.

FIELD OF THE INVENTION

The present invention relates to therapeutic use of immunoconjugates ofantibodies or antigen-binding antibody fragments and camptothecins, suchas SN-38, with improved ability to target various cancer cells in humansubjects. In preferred embodiments, the antibodies and therapeuticmoieties are linked via an intracellularly-cleavable linkage thatincreases therapeutic efficacy. In more preferred embodiments, theimmunoconjugates are administered at specific dosages and/or specificschedules of administration that optimize the therapeutic effect. Theoptimized dosages and schedules of administration of SN-38-conjugatedantibodies for human therapeutic use disclosed herein show unexpectedsuperior efficacy that could not have been predicted from animal modelstudies, allowing effective treatment of cancers that are resistant tostandard anti-cancer therapies, including the parental compound,irinotecan (CPT-11).

BACKGROUND OF THE INVENTION

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer in humans, andvirtually no application in other diseases, such as infectious andautoimmune diseases. The toxic agent is most commonly a chemotherapeuticdrug, although particle-emitting radionuclides, or bacterial or planttoxins, have also been conjugated to MAbs, especially for the therapy ofcancer (Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-243) and, more recently, with radioimmunoconjugates for thepreclinical therapy of certain infectious diseases (Dadachova andCasadevall, Q J Nucl Med Mol Imaging 2006; 50(3): 193-204).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using verywell-defined conjugation chemistries, often at specific sites remotefrom the MAbs' antigen binding regions; (c) MAb-chemotherapeutic drugconjugates can be made more reproducibly and usually with lessimmunogenicity than chemical conjugates involving MAbs and bacterial orplant toxins, and as such are more amenable to commercial developmentand regulatory approval; and (d) the MAb-chemotherapeutic drugconjugates are orders of magnitude less toxic systemically thanradionuclide MAb conjugates, particularly to the radiation-sensitivebone marrow.

Camptothecin (CPT) and its derivatives are a class of potent antitumoragents. Irinotecan (also referred to as CPT-11) and topotecan are CPTanalogs that are approved cancer therapeutics (Iyer and Ratain, CancerChemother. Phamacol. 42: S31-S43 (1998)). CPTs act by inhibitingtopoisomerase I enzyme by stabilizing topoisomerase I-DNA complex (Liu,et al. in The Camptothecins: Unfolding Their Anticancer Potential, LiehrJ. G., Giovanella, B. C. and Verschraegen (eds), NY Acad Sci., NY922:1-10 (2000)). CPTs present specific issues in the preparation ofconjugates. One issue is the insolubility of most CPT derivatives inaqueous buffers. Second, CPTs provide specific challenges for structuralmodification for conjugating to macromolecules. For instance, CPT itselfcontains only a tertiary hydroxyl group in ring-E. The hydroxylfunctional group in the case of CPT must be coupled to a linker suitablefor subsequent protein conjugation; and in potent CPT derivatives, suchas SN-38, the active metabolite of the chemotherapeutic CPT-11, andother C-10-hydroxyl-containing derivatives such as topotecan and10-hydroxy-CPT, the presence of a phenolic hydroxyl at the C-10 positioncomplicates the necessary C-20-hydroxyl derivatization. Third, thelability under physiological conditions of the δ-lactone moiety of theE-ring of camptothecins results in greatly reduced antitumor potency.Therefore, the conjugation protocol is performed such that it is carriedout at a pH of 7 or lower to avoid the lactone ring opening. However,conjugation of a bifunctional CPT possessing an amine-reactive groupsuch as an active ester would typically require a pH of 8 or greater.Fourth, an intracellularly-cleavable moiety preferably is incorporatedin the linker/spacer connecting the CPTs and the antibodies or otherbinding moieties.

A need exists for more effective methods of preparing and administeringantibody-CPT conjugates, such as antibody-SN-38 conjugates. Preferably,the methods comprise optimized dosing and administration schedules thatmaximize efficacy and minimize toxicity of the antibody-CPT conjugatesfor therapeutic use in human patients.

SUMMARY OF THE INVENTION

As used herein, the abbreviation “CPT” may refer to camptothecin or anyof its derivatives, such as SN-38, unless expressly stated otherwise.The present invention resolves an unfulfilled need in the art byproviding improved methods and compositions for preparing andadministering CPT-antibody immunoconjugates. Preferably, thecamptothecin is SN-38. The disclosed methods and compositions are of usefor the treatment of a variety of diseases and conditions which arerefractory or less responsive to other forms of therapy, and can includediseases against which suitable antibodies or antigen-binding antibodyfragments for selective targeting can be developed, or are available orknown. Preferred diseases or conditions that may be treated with thesubject immunoconjugates include, for example, cancer or diseases causedby infectious organisms.

Preferably, the targeting moiety is an antibody, antibody fragment,bispecific or other multivalent antibody, or other antibody-basedmolecule or compound. The antibody can be of various isotypes,preferably human IgG1, IgG2, IgG3 or IgG4, more preferably comprisinghuman IgG1 hinge and constant region sequences. The antibody or fragmentthereof can be a chimeric human-mouse, a chimeric human-primate, ahumanized (human framework and murine hypervariable (CDR) regions), orfully human antibody, as well as variations thereof, such as half-IgG4antibodies (referred to as “unibodies”), as described by van der NeutKolfschoten et al. (Science 2007; 317:1554-1557). More preferably, theantibody or fragment thereof may be designed or selected to comprisehuman constant region sequences that belong to specific allotypes, whichmay result in reduced immunogenicity when the immunoconjugate isadministered to a human subject. Preferred allotypes for administrationinclude a non-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 orG1m3,1,2. More preferably, the allotype is selected from the groupconsisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

Antibodies of use may bind to any disease-associated antigen known inthe art. Where the disease state is cancer, for example, many antigensexpressed by or otherwise associated with tumor cells are known in theart, including but not limited to, carbonic anhydrase IX,alpha-fetoprotein (AFP), α-actinin-4, A3, antigen specific for A33antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1,CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A,CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83,CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A,CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp),CEA (CEACAM5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2),EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3,folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growthfactor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT,macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1,MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90,PAM4 antigen, pancreatic cancer mucin, PD-1 receptor, placental growthfactor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAIVIE, PSMA,P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100,survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tnantigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR,ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b,C5a, C5, an angiogenesis marker, bcl-2, bcl-6, Kras, an oncogene markerand an oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207). Preferably, theantibody binds to CEACAM5, CEACAM6, EGP-1 (TROP-2), MUC-16, AFP,MUC5a,c, PAM4 antigen, CD74, CD19, CD20, CD22 or HLA-DR.

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. patent application Ser. No. 12/722,645, filedMar. 12, 2010), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496),the Examples section of each cited patent or application incorporatedherein by reference. More preferably, the antibody is IMMU-31(anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti-CEACAM5), hMN-3(anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1 (anti-CD74), hLL2(anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20(anti-CD20). As used herein, the terms epratuzumab and hLL2 areinterchangeable, as are the terms veltuzumab and hA20, hL243 g4P,hL243gamma4P and IMMU-114.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4),abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab(anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101,anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), Benlysta (Human GenomeSciences); antibodies for therapy of Alzheimer's disease such as Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,solanezumab and infliximab; anti-fibrin antibodies like 59D8, T2G1s,MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202(Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson & Johnson);(anti-HIV antibodies such as P4/D10 (U.S. Pat. No. 8,333,971), Ab 75, Ab76, Ab 77 (Paulik et al., 1999, Biochem Pharmacol 58:1781-90), as wellas the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. No. 5,831,034, U.S. Pat. No.5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos etal., Antimicrob. Agents Chemother. 2006; 50(5):1773-9, all incorporatedherein by reference; and antibodies against pathogens such as CR6261(anti-influenza), exbivirumab (anti-hepatitis B), felvizumab(anti-respiratory syncytial virus), foravirumab (anti-rabies virus),motavizumab (anti-respiratory syncytial virus), palivizumab(anti-respiratory syncytial virus), panobacumab (anti-Pseudomonas),rafivirumab (anti-rabies virus), regavirumab (anti-cytomegalovirus),sevirumab (anti-cytomegalovirus), tivirumab (anti-hepatitis B), andurtoxazumab (anti-E. coli).

In a preferred embodiment, the chemotherapeutic moiety is selected fromcamptothecin (CPT) and its analogs and derivatives and is morepreferably SN-38. However, other chemotherapeutic moieties that may beutilized include taxanes (e.g, baccatin III, taxol), epothilones,anthracyclines (e.g., doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolinodoxorubicin (2-PDOX) or a prodrug formof 2-PDOX (pro-2-PDOX); see, e.g., Priebe W (ed.), ACS symposium series574, published by American Chemical Society, Washington D.C., 1995 (332pp) and Nagy et al., Proc. Natl. Acad. Sci. USA 93:2464-2469, 1996),benzoquinoid ansamycins exemplified by geldanamycin (DeBoer et al.,Journal of Antibiotics 23:442-447, 1970; Neckers et al., Invest. NewDrugs 17:361-373, 1999), and the like. Preferably, the antibody orfragment thereof links to at least one chemotherapeutic moiety;preferably 1 to about 5 chemotherapeutic moieties; more preferably 6 ormore chemotherapeutic moieties, most preferably about 6 to about 12chemotherapeutic moieties.

An example of a water soluble CPT derivative is CPT-11. Extensiveclinical data are available concerning CPT-11's pharmacology and its invivo conversion to the active SN-38 (Iyer and Ratain, Cancer ChemotherPharmacol. 42:S31-43 (1998); Mathijssen et al., Clin Cancer Res.7:2182-2194 (2002); Rivory, Ann NY Acad Sci. 922:205-215, 2000)). Theactive form SN-38 is about 2 to 3 orders of magnitude more potent thanCPT-11. In specific preferred embodiments, the immunoconjugate may be anhMN-14-SN-38, hMN-3-SN-38, hMN-15-SN-38, IMMU-31-SN-38, hRS7-SN-38,hA20-SN-38, hL243-SN-38, hLL1-SN-38 or hLL2-SN-38 conjugate.

Various embodiments may concern use of the subject methods andcompositions to treat a cancer, including but not limited tonon-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias,Burkitt lymphoma, Hodgkin's lymphoma, acute large B-cell lymphoma, hairycell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, T-cell lymphomas andleukemias, multiple myeloma, Waldenstrom's macroglobulinemia,carcinomas, melanomas, sarcomas, gliomas, bone, and skin cancers. Thecarcinomas may include carcinomas of the oral cavity, esophagus,gastrointestinal tract, pulmonary tract, lung, stomach, colon, breast,ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas,bone, brain, connective tissue, liver, gall bladder, urinary bladder,kidney, skin, central nervous system and testes.

In addition, the subject methods and compositions may be used to treatan infectious disease, for example diseases involving infection bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi,viruses, parasites, or other microbial agents. Examples include humanimmunodeficiency virus (HIV) causing AIDS, Mycobacterium oftuberculosis, Streptococcus agalactiae, methicillin-resistantStaphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes,Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis,Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum,Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes,West Nile virus, Pseudomonas aeruginosa, Mycobacterium leprae, Brucellaabortus, rabies virus, influenza virus, cytomegalovirus, herpes simplexvirus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, reo virus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodiumvivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi,Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orate, M. arginini, Acholeplasma laidlawii, M. salivarium and M.pneumoniae. A review listing antibodies against infectious organisms(antitoxin and antiviral antibodies), as well as other targets, iscontained in Casadevall, Clin Immunol 1999; 93(1):5-15, incorporatedherein by reference.

In certain embodiments involving treatment of cancer, the drugconjugates may be used in combination with surgery, radiation therapy,chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy,immunomodulators, vaccines, and the like. These combination therapiescan allow lower doses of each therapeutic to be given in suchcombinations, thus reducing certain severe side effects, and potentiallyreducing the courses of therapy required. When there is no or minimaloverlapping toxicity, ful doses of each can also be given.

In infectious diseases, the drug immunoconjugates can be combined withother therapeutic drugs, immunomodulators, naked MAbs, or vaccines(e.g., MAbs against hepatitis, HIV, or papilloma viruses, or vaccinesbased on immunogens of these viruses, or kinase inhibitors, such as inhepatitis B). Antibodies and antigen-based vaccines against these andother viral pathogens are known in the art and, in some cases, alreadyin commercial use. The development of anti-infective monoclonalantibodies has been reviewed recently by Reichert and Dewitz (Nat RevDrug Discovery 2006; 5:191-195), incorporated herein by reference, whichsummarizes the priority pathogens against which naked antibody therapyhas been pursued, resulting in only 2 pathogens against which antibodiesare either in Phase III clinical trials or are being marketed(respiratory syncytial virus and methicillin-resistant Staphylococcusaureus), with 25 others in clinical studies and 20 discontinued duringclinical study. For combination therapy, the use of radioimmunotherapyfor the treatment of infectious organisms is disclosed, for example, inU.S. Pat. Nos. 4,925,648; 5,332,567; 5,439,665; 5,601,825; 5,609,846;5,612,016; 6,120,768; 6,319,500; 6,458,933; 6,548,275; and in U.S.Patent Application Publication Nos. 20020136690 and 20030103982, theExamples section of each of which is incorporated herein by reference.

Preferred optimal dosing of immunoconjugates may include a dosage ofbetween 3 mg/kg and 20 mg/kg, preferably given either weekly, twiceweekly or every other week. The optimal dosing schedule may includetreatment cycles of two consecutive weeks of therapy followed by one,two, three or four weeks of rest, or alternating weeks of therapy andrest, or one week of therapy followed by two, three or four weeks ofrest, or three weeks of therapy followed by one, two, three or fourweeks of rest, or four weeks of therapy followed by one, two, three orfour weeks of rest, or five weeks of therapy followed by one, two,three, four or five weeks of rest, or administration once every twoweeks, once every three weeks or once a month. Treatment may be extendedfor any number of cycles, preferably at least 2, at least 4, at least 6,at least 8, at least 10, at least 12, at least 14, or at least 16cycles. The dosage may be up to 24 mg/kg. Exemplary dosages of use mayinclude 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24mg/kg. Preferred dosages are 4, 6, 8, 9, 10, 12, 14, 16 or 18 mg/kg. Theperson of ordinary skill will realize that a variety of factors, such asage, general health, specific organ function or weight, as well aseffects of prior therapy on specific organ systems (e.g., bone marrow)may be considered in selecting an optimal dosage of immunoconjugate, andthat the dosage and/or frequency of administration may be increased ordecreased during the course of therapy. The dosage may be repeated asneeded, with evidence of tumor shrinkage observed after as few as 4 to 8doses. The optimized dosages and schedules of administration disclosedherein show unexpected superior efficacy and reduced toxicity in humansubjects, which could not have been predicted from animal model studies.Surprisingly, the superior efficacy allows treatment of tumors that werepreviously found to be resistant to one or more standard anti-cancertherapies, including the parental compound, CPT-11, from which SN-38 isderived in vivo.

The subject methods may include use of CT and/or PET/CT, or Mill, tomeasure tumor response at regular intervals. Blood levels of tumormarkers, such as CEA (carcinoembryonic antigen), CA19-9, AFP, CA 15.3,or PSA, may also be monitored. Dosages and/or administration schedulesmay be adjusted as needed, according to the results of imaging and/ormarker blood levels.

A surprising result with the instant claimed compositions and methods isthe unexpected tolerability of high doses of antibody-drug conjugate,even with repeated infusions, with only relatively low-grade toxicitiesof nausea and vomiting observed, or manageable neutropenia. A furthersurprising result is the lack of accumulation of the antibody-drugconjugate, unlike other products that have conjugated SN-38 to albumin,PEG or other carriers. The lack of accumulation is associated withimproved tolerability and lack of serious toxicity even after repeatedor increased dosing. These surprising results allow optimization ofdosage and delivery schedule, with unexpectedly high efficacies and lowtoxicities. The claimed methods provide for shrinkage of solid tumors,in individuals with previously resistant cancers, of 15% or more,preferably 20% or more, preferably 30% or more, more preferably 40% ormore in size (as measured by longest diameter). The person of ordinaryskill will realize that tumor size may be measured by a variety ofdifferent techniques, such as total tumor volume, maximal tumor size inany dimension or a combination of size measurements in severaldimensions. This may be with standard radiological procedures, such ascomputed tomography, ultrasonography, and/or positron-emissiontomography. The means of measuring size is less important than observinga trend of decreasing tumor size with immunoconjugate treatment,preferably resulting in elimination of the tumor.

While the immunoconjugate may be administered as a periodic bolusinjection, in alternative embodiments the immunoconjugate may beadministered by continuous infusion of antibody-drug conjugates. Inorder to increase the Cmax and extend the PK of the immunoconjugate inthe blood, a continuous infusion may be administered for example byindwelling catheter. Such devices are known in the art, such asHICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see, e.g., Skolnik et al.,Ther Drug Monit 32:741-48, 2010) and any such known indwelling cathetermay be used. A variety of continuous infusion pumps are also known inthe art and any such known infusion pump may be used. The dosage rangefor continuous infusion may be between 0.1 and 3.0 mg/kg per day. Morepreferably, these immunoconjugates can be administered by intravenousinfusions over relatively short periods of 2 to 5 hours, more preferably2-3 hours.

In particularly preferred embodiments, the immunoconjugates and dosingschedules may be efficacious in patients resistant to standardtherapies. For example, an hMN-14-SN-38 immunoconjugate may beadministered to a patient who has not responded to prior therapy withirinotecan, the parent agent of SN-38. Surprisingly, theirinotecan-resistant patient may show a partial or even a completeresponse to hMN-14-SN-38. The ability of the immunoconjugate tospecifically target the tumor tissue may overcome tumor resistance byimproved targeting and enhanced delivery of the therapeutic agent.Alternatively, an anti-CEACAM5 immunoconjugate, such as hMN-14, may beco-administered with an anti-CEACAM6 immunoconjugate, such as hMN-3 orhMN-15. Other antibody-SN-38 immunoconjugates may show similar improvedefficacy and/or decreased toxicity, compared to alternative standardtherapeutic treatments, and combinations of different SN-38immunoconjugates, or SN-38-antibody conjugates in combination with anantibody conjugated to a radionuclide, toxin or other drug, may provideeven more improved efficacy and/or reduced toxicity. A specificpreferred subject may be a metastatic colon cancer patient, atriple-negative breast cancer patient, a HER+, ER+, progesterone+breastcancer patient, a metastatic non-small-cell lung cancer (NSCLC) patient,a metastatic pancreatic cancer patient, a metastatic renal cellcarcinoma patient, a metastatic gastric cancer patient, a metastaticprostate cancer patient, or a metastatic small-cell lung cancer patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. In vivo therapy of athymic nude mice, bearing Capan 1 humanpancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 2. In vivo therapy of athymic nude mice, bearing BxPC3 humanpancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 3. In vivo therapy of athymic nude mice, bearing LS174T human coloncarcinoma, with hMN-14-CL2A-SN-38 conjugate.

FIG. 4. Survival curves of hMN14-CL-SN-38 treated mice bearing GW-39lung metastatic disease.

FIG. 5A. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humannon-small cell lung tumor xenografts. Mice bearing Calu-3 tumors (N=5-7)were injected with hRS7-CL2-SN-38 every 4 days for a total of 4injections (q4dx4). All the ADCs and controls were administered in theamounts indicated (expressed as amount of SN-38 per dose; longarrows=conjugate injections, short arrows=irinotecan injections).

FIG. 5B. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humancolorectal tumor xenografts. COLO 205 tumor-bearing mice (N=5) wereinjected 8 times (q4dx8) with the ADC or every 2 days for a total of 5injections (q2dx5) with the MTD of irinotecan. All the ADCs and controlswere administered in the amounts indicated (expressed as amount of SN-38per dose; long arrows=conjugate injections, short arrows=irinotecaninjections).

FIG. 5C. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humanpancreatic cancer xenografts. Capan-1 (N=10) tumor-bearing mice (N=10)were treated twice weekly for 4 weeks with the agents indicated. All theADCs and controls were administered in the amounts indicated (expressedas amount of SN-38 per dose; long arrows=conjugate injections, shortarrows=irinotecan injections).

FIG. 5D. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humanpancreatic cancer xenografts. BxPC-3 tumor-bearing mice (N=10) weretreated twice weekly for 4 weeks with the agents indicated. All the ADCsand controls were administered in the amounts indicated (expressed asamount of SN-38 per dose; long arrows=conjugate injections, shortarrows=irinotecan injections).

FIG. 5E. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humansquamous cell lung carcinoma xenografts. In addition to ADC given twiceweekly for 4 week, SK-MES-1 tumor-bearing (N=8) mice received the MTD ofCPT-11 (q2dx5). All the ADCs and controls were administered in theamounts indicated (expressed as amount of SN-38 per dose; longarrows=conjugate injections, short arrows=irinotecan injections).

FIG. 6. Comparative efficacy of epratuzumab (Emab)-SN-38 and veltuzumab(Vmab)-SN-38 conjugates in the subcutaneous Ramos model. Nude mice (N=10per group) with tumors averaging approximately 0.35 cm³ (0.20-0.55 cm³)were administered 0.25 or 0.5 mg of each conjugate twice weekly for 4weeks.

FIG. 7A. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 75 μg of each conjugate per dose (54.5 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel. C,survival curves (solid gray) for another group of animals given weeklyintraperitoneal injections of irinotecan (6.5 μg/dose; SN-38 equivalentsapproximately the same as the 250-μg dose of the Emab-SN-38 conjugate).

FIG. 7B. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 125 μg of each conjugate per dose (91 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel. C,survival curves (solid gray) for another group of animals given weeklyintraperitoneal injections of irinotecan (6.5 μg/dose; SN-38 equivalentsapproximately the same as the 250-μg dose of the Emab-SN-38 conjugate).

FIG. 7C. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 250 μg of each conjugate per dose (182 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel. Survivalcurves (solid gray) are also shown for another group of animals givenweekly intraperitoneal injections of irinotecan (6.5 μg/dose; SN-38equivalents approximately the same as the 250-μg dose of the Emab-SN-38conjugate).

FIG. 8. History of prior treatment of patient, before administeringIMMU-130 (labetuzumab-NS-38). Prior treatment included stage IV CRCcoloectomy/hepatectomy (partial lobe), radiofrequency ablation therapyof liver metasteses, wedge resection of lung metasteses, andchemotherapy with irinotecan/oxaliplatin, Folfirinox,Folfirinox+bevacizumab, bevacizumab+5-FU/leucovorin, FolFiri,Folfiri+cetuximab, and cetuximab alone. The patient received doses of 16mg/kg of IMMU-132 by slow IV infusion every other week for a total of 17treatment doses.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes. As usedbelow, the abbreviation “MAb” may be used interchangeably to refer to anantibody, antibody fragment, monoclonal antibody or multispecificantibody.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317(14September):1554-1557). Regardless of structure, an antibody fragment ofuse binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). The fragments may be constructed in different ways toyield multivalent and/or multispecific binding forms.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. A naked antibody may exhibit therapeutic and/orcytotoxic effects, for example by Fc-dependent functions, such ascomplement fixation (CDC) and ADCC (antibody-dependent cellcytotoxicity). However, other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,inhibition of heterotypic or homotypic adhesion, and interference insignaling pathways, may also provide a therapeutic effect. Nakedantibodies include polyclonal and monoclonal antibodies, naturallyoccurring or recombinant antibodies, such as chimeric, humanized orhuman antibodies and fragments thereof. In some cases a “naked antibody”may also refer to a “naked” antibody fragment. As defined herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to a therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, human antibody variable domain genes arecloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, the Examples section of each of which is incorporatedherein by reference.

A therapeutic agent is an atom, molecule, or compound that is useful inthe treatment of a disease. Examples of therapeutic agents include, butare not limited to, antibodies, antibody fragments, immunoconjugates,drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases(including DNAses and RNAses), hormones, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radionuclides,oligonucleotides, interference RNA, siRNA, RNAi, anti-angiogenic agents,chemotherapeutic agents, cyokines, chemokines, prodrugs, enzymes,binding proteins or peptides or combinations thereof.

An immunoconjugate is an antibody, antigen-binding antibody fragment,antibody complex or antibody fusion protein that is conjugated to atherapeutic agent. Conjugation may be covalent or non-covalent.Preferably, conjugation is covalent.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat is released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which acts as an intercellularmediator between cells. As the skilled artisan will understand, examplesof cytokines include lymphokines, monokines, interleukins, and severalrelated signaling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation. Exemplary interferons include interferon-α,interferon-β, interferon-γ and interferon-λ.

CPT is an abbreviation for camptothecin, and as used in the presentapplication CPT represents camptothecin itself or an analog orderivative of camptothecin, such as SN-38. The structures ofcamptothecin and some of its analogs, with the numbering indicated andthe rings labeled with letters A-E, are given in formula 1 in Chart 1below.

Camptothecin Conjugates

Non-limiting methods and compositions for preparing immunoconjugatescomprising a camptothecin therapeutic agent attached to an antibody orantigen-binding antibody fragment are described below. In preferredembodiments, the solubility of the drug is enhanced by placing a definedpolyethyleneglycol (PEG) moiety (i.e., a PEG containing a defined numberof monomeric units) between the drug and the antibody, wherein thedefined PEG is a low molecular weight PEG, preferably containing 1-30monomeric units, more preferably containing 1-12 monomeric units.

Preferably, a first linker connects the drug at one end and mayterminate with an acetylene or an azide group at the other end. Thisfirst linker may comprise a defined PEG moiety with an azide oracetylene group at one end and a different reactive group, such ascarboxylic acid or hydroxyl group, at the other end. Said bifunctionaldefined PEG may be attached to the amine group of an amino alcohol, andthe hydroxyl group of the latter may be attached to the hydroxyl groupon the drug in the form of a carbonate. Alternatively, the non-azide (oracetylene) moiety of said defined bifunctional PEG is optionallyattached to the N-terminus of an L-amino acid or a polypeptide, with theC-terminus attached to the amino group of amino alcohol, and the hydroxygroup of the latter is attached to the hydroxyl group of the drug in theform of carbonate or carbamate, respectively.

A second linker, comprising an antibody-coupling group and a reactivegroup complementary to the azide (or acetylene) group of the firstlinker, namely acetylene (or azide), may react with the drug-(firstlinker) conjugate via acetylene-azide cycloaddition reaction to furnisha final bifunctional drug product that is useful for conjugating todisease-targeting antibodies. The antibody-coupling group is preferablyeither a thiol or a thiol-reactive group.

Methods for selective regeneration of the 10-hydroxyl group in thepresence of the C-20 carbonate in preparations of drug-linker precursorinvolving CPT analogs such as SN-38 are provided below. Other protectinggroups for reactive hydroxyl groups in drugs such as the phenolichydroxyl in SN-38, for example t-butyldimethylsilyl ort-butyldiphenylsilyl, may also be used, and these are deprotected bytetrabutylammonium fluoride prior to linking of the derivatized drug toan antibody-coupling moiety. The 10-hydroxyl group of CPT analogs isalternatively protected as an ester or carbonate, other than ‘BOC’, suchthat the bifunctional CPT is conjugated to an antibody without priordeprotection of this protecting group. The protecting group is readilydeprotected under physiological pH conditions after the bioconjugate isadministered.

In the acetylene-azide coupling, referred to as ‘click chemistry’, theazide part may be on L2 with the acetylene part on L3. Alternatively, L2may contain acetylene, with L3 containing azide. ‘Click chemistry’refers to a copper (+1)-catalyzed cycloaddition reaction between anacetylene moiety and an azide moiety (Kolb H C and Sharpless K B, DrugDiscov Today 2003; 8: 1128-37), although alternative forms of clickchemistry are known and may be used. Click chemistry takes place inaqueous solution at near-neutral pH conditions, and is thus amenable fordrug conjugation. The advantage of click chemistry is that it ischemoselective, and complements other well-known conjugation chemistriessuch as the thiol-maleimide reaction.

While the present application focuses on use of antibodies or antibodyfragments as targeting moieties, the skilled artisan will realize thatwhere a conjugate comprises an antibody or antibody fragment, anothertype of targeting moiety, such as an aptamer, avimer, affibody orpeptide ligand, may be substituted.

An exemplary preferred embodiment is directed to a conjugate of a drugderivative and an antibody of the general formula 2,

MAb-[L2]-[L1]-[AA]_(m)[A′]-Drug  (2)

where MAb is a disease-targeting antibody; L2 is a component of thecross-linker comprising an antibody-coupling moiety and one or more ofacetylene (or azide) groups; L1 comprises a defined PEG with azide (oracetylene) at one end, complementary to the acetylene (or azide) moietyin L2, and a reactive group such as carboxylic acid or hydroxyl group atthe other end; AA is an L-amino acid; m is an integer with values of 0,1, 2, 3, or 4; and A′ is an additional spacer, selected from the groupof ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, orsubstituted or unsubstituted ethylenediamine. The L amino acids of ‘AA’are selected from alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. If the A′ group contains hydroxyl, itis linked to the hydroxyl group or amino group of the drug in the formof a carbonate or carbamate, respectively.

In a preferred embodiment of formula 2, A′ is a substituted ethanolaminederived from an L-amino acid, wherein the carboxylic acid group of theamino acid is replaced by a hydroxymethyl moiety. A′ may be derived fromany one of the following L-amino acids: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine.

In an example of the conjugate of the preferred embodiment of formula 2,m is 0, A′ is L-valinol, and the drug is exemplified by SN-38. Theresultant structure is shown in formula 3.

In another example of the conjugate of the preferred embodiment offormula 2, m is 1 and represented by a derivatized L-lysine, A′ isL-valinol, and the drug is exemplified by SN-38. The structure is shownin formula 4.

In this embodiment, an amide bond is first formed between the carboxylicacid of an amino acid such as lysine and the amino group of valinol,using orthogonal protecting groups for the lysine amino groups. Theprotecting group on the N-terminus of lysine is removed, keeping theprotecting group on the side chain of lysine intact, and the N-terminusis coupled to the carboxyl group on the defined PEG with azide (oracetylene) at the other end. The hydroxyl group of valinol is thenattached to the 20-chloroformate derivative of 10-hydroxy-protectedSN-38, and this intermediate is coupled to an L2 component carrying theantibody-binding moiety as well as the complementary acetylene (orazide) group involved in the click cycloaddition chemistry. Finally,removal of protecting groups at both lysine side chain and SN-38 givesthe product of this example, shown in formula 3.

While not wishing to be bound by theory, the small MW SN-38 product,namely valinol-SN-38 carbonate, generated after intracellularproteolysis, has the additional pathway of liberation of intact SN-38through intramolecular cyclization involving the amino group of valinoland the carbonyl of the carbonate.

In another preferred embodiment, A′ of the general formula 2 is A-OH,whereby A-OH is a collapsible moiety such as 4-aminobenzyl alcohol or asubstituted 4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkyl groupat the benzylic position, and the latter, via its amino group, isattached to an L-amino acid or a polypeptide comprising up to fourL-amino acid moieties; wherein the N-terminus is attached to across-linker terminating in the antibody-binding group.

An example of a preferred embodiment is given below, wherein the A-OHembodiment of A′ of general formula (2) is derived from substituted4-aminobenzyl alcohol, and ‘AA’ is comprised of a single L-amino acidwith m=1 in the general formula (2), and the drug is exemplified withSN-38. The structure is represented below (formula 5, referred to asMAb-CLX-SN-38). Single amino acid of AA is selected from any one of thefollowing L-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. The substituent R on 4-aminobenzylalcohol moiety (A-OH embodiment of A′) is hydrogen or an alkyl groupselected from C1-C10 alkyl groups.

An embodiment of MAb-CLX-SN-38 of formula 5, wherein the single aminoacid AA is L-lysine and R═H, and the drug is exemplified by SN-38(formula 6; referred to as MAb-CL2A-SN-38).

Other embodiments are possible within the context of10-hydroxy-containing camptothecins, such as SN-38. In the example ofSN-38 as the drug, the more reactive 10-hydroxy group of the drug isderivatized leaving the 20-hydroxyl group unaffected. Within the generalformula 2, A′ is a substituted ethylenediamine. An example of thisembodiment is represented by the formula ‘7’ below, wherein the phenolichydroxyl group of SN-38 is derivatized as a carbamate with a substitutedethylenediamine, with the other amine of the diamine derivatized as acarbamate with a 4-aminobenzyl alcohol, and the latter's amino group isattached to Phe-Lys dipeptide. In this structure (formula 7), R and R′are independently hydrogen or methyl. It is referred to asMAb-CL17-SN-38 or MAb-CL2E-SN-38, when R═R′=methyl.

In a preferred embodiment, AA comprises a polypeptide moiety, preferablya di, tri or tetrapeptide, that is cleavable by intracellular peptidase.Examples are: Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu (Trouet et al.,1982).

In another preferred embodiment, the L1 component of the conjugatecontains a defined polyethyleneglycol (PEG) spacer with 1-30 repeatingmonomeric units. In a further preferred embodiment, PEG is a defined PEGwith 1-12 repeating monomeric units. The introduction of PEG may involveusing heterobifunctionalized PEG derivatives which are availablecommercially. The heterobifunctional PEG may contain an azide oracetylene group. An example of a heterobifunctional defined PEGcontaining 8 repeating monomeric units, with ‘NHS’ being succinimidyl,is given below in formula 8:

In a preferred embodiment, L2 has a plurality of acetylene (or azide)groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5,and a single antibody-binding moiety.

A representative SN-38 conjugate of an antibody containing multiple drugmolecules and a single antibody-binding moiety is shown below. The ‘L2’component of this structure is appended to 2 acetylenic groups,resulting in the attachment of two azide-appended SN-38 molecules. Thebonding to MAb is represented as a succinimide.

-   -   Where R residue is:

In preferred embodiments, when the bifunctional drug contains athiol-reactive moiety as the antibody-binding group, the thiols on theantibody are generated on the lysine groups of the antibody using athiolating reagent. Methods for introducing thiol groups onto antibodiesby modifications of MAb's lysine groups are well known in the art (Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). Alternatively, mild reduction ofinterchain disulfide bonds on the antibody (Willner et al., BioconjugateChem. 4:521-527 (1993)) using reducing agents such as dithiothreitol(DTT) can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple drug moieties in the interchainregion of the MAb away from the antigen-binding region. In a morepreferred embodiment, attachment of SN-38 to reduced disulfidesulfhydryl groups results in formation of an antibody-SN-38immunoconjugate with 6 SN-38 moieties covalently attached per antibodymolecule. Other methods of providing cysteine residues for attachment ofdrugs or other therapeutic agents are known, such as the use of cysteineengineered antibodies (see U.S. Pat. No. 7,521,541, the Examples sectionof which is incorporated herein by reference.)

In alternative preferred embodiments, the chemotherapeutic moiety isselected from the group consisting of doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), Pro-2PDOX, CPT,10-hydroxy camptothecin, SN-38, topotecan, lurtotecan,9-aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin,ansamycins, and epothilones. In a more preferred embodiment, thechemotherapeutic moiety is SN-38. Preferably, in the conjugates of thepreferred embodiments, the antibody links to at least onechemotherapeutic moiety; preferably 1 to about 12 chemotherapeuticmoieties; most preferably about 6 to about 12 chemotherapeutic moieties.

Furthermore, in a preferred embodiment, the linker component ‘L2’comprises a thiol group that reacts with a thiol-reactive residueintroduced at one or more lysine side chain amino groups of saidantibody. In such cases, the antibody is pre-derivatized with athiol-reactive group such as a maleimide, vinylsulfone, bromoacetamide,or iodoacetamide by procedures well described in the art.

In the context of this work, a process was surprisingly discovered bywhich CPT drug-linkers can be prepared wherein CPT additionally has a10-hydroxyl group. This process involves, but is not limited to, theprotection of the 10-hydroxyl group as a t-butyloxycarbonyl (BOC)derivative, followed by the preparation of the penultimate intermediateof the drug-linker conjugate. Usually, removal of BOC group requirestreatment with strong acid such as trifluoroacetic acid (TFA). Underthese conditions, the CPT 20-O-linker carbonate, containing protectinggroups to be removed, is also susceptible to cleavage, thereby givingrise to unmodified CPT. In fact, the rationale for using a mildlyremovable methoxytrityl (MMT) protecting group for the lysine side chainof the linker molecule, as enunciated in the art, was precisely to avoidthis possibility (Walker et al., 2002). It was discovered that selectiveremoval of phenolic BOC protecting group is possible by carrying outreactions for short durations, optimally 3-to-5 minutes. Under theseconditions, the predominant product was that in which the ‘BOC’ at10-hydroxyl position was removed, while the carbonate at ‘20’ positionwas intact.

An alternative approach involves protecting the CPT analog's 10-hydroxyposition with a group other than ‘BOC’, such that the final product isready for conjugation to antibodies without a need for deprotecting the10-OH protecting group. The 10-hydroxy protecting group, which convertsthe 10-OH into a phenolic carbonate or a phenolic ester, is readilydeprotected by physiological pH conditions or by esterases after in vivoadministration of the conjugate. The faster removal of a phenoliccarbonate at the 10 position vs. a tertiary carbonate at the 20 positionof 10-hydroxycamptothecin under physiological condition has beendescribed by He et al. (He et al., Bioorganic & Medicinal Chemistry 12:4003-4008 (2004)). A 10-hydroxy protecting group on SN-38 can be ‘COR’where R can be a substituted alkyl such as “N(CH₃)₂—(CH₂)_(n)—” where nis 1-10 and wherein the terminal amino group is optionally in the formof a quaternary salt for enhanced aqueous solubility, or a simple alkylresidue such as “CH₃—(CH₂)_(n)—” where n is 0-10, or it can be an alkoxymoiety such as “CH₃—(CH₂)_(n)—O—” where n is 0-10, or“N(CH₃)₂—(CH₂)_(n)—O—” where n is 2-10, or“R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl or methyl and n is aninteger with values of 0-10. These 10-hydroxy derivatives are readilyprepared by treatment with the chloroformate of the chosen reagent, ifthe final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-X are as described in earlier sections, thebifunctional drug moiety, [L2]-[L1]-[AA]_(m)-[A-X]-Drug is firstprepared, followed by the conjugation of the bifunctional drug moiety tothe antibody (indicated herein as “MAb”).

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-OH are as described in earlier sections,the bifunctional drug moiety is prepared by first linking A-OH to theC-terminus of AA via an amide bond, followed by coupling the amine endof AA to a carboxylic acid group of L1. If AA is absent (i.e. m=0), A-OHis directly attached to L1 via an amide bond. The cross-linker,[L1]-[AA]_(m)-[A-OH], is attached to drug's hydroxyl or amino group, andthis is followed by attachment to the L1 moiety, by taking recourse tothe reaction between azide (or acetylene) and acetylene (or azide)groups in L1 and L2 via click chemistry.

In one embodiment, the antibody is a monoclonal antibody (MAb). In otherembodiments, the antibody may be a multivalent and/or multispecific MAb.The antibody may be a murine, chimeric, humanized, or human monoclonalantibody, and said antibody may be in intact, fragment (Fab, Fab′,F(ab)₂, F(ab′)₂), or sub-fragment (single-chain constructs) form, or ofan IgG1, IgG2a, IgG3, IgG4, IgA isotype, or submolecules therefrom.

In a preferred embodiment, the antibody binds to an antigen or epitopeof an antigen expressed on a cancer or malignant cell. The cancer cellis preferably a cell from a hematopoietic tumor, carcinoma, sarcoma,melanoma or a glial tumor. A preferred malignancy to be treatedaccording to the present invention is a malignant solid tumor orhematopoietic neoplasm.

In a preferred embodiment, the intracellularly-cleavable moiety may becleaved after it is internalized into the cell upon binding by theMAb-drug conjugate to a receptor thereof, and particularly cleaved byesterases and peptidases.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Köhler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

The skilled artisan will realize that the claimed methods andcompositions may utilize any of a wide variety of antibodies known inthe art. Antibodies of use may be commercially obtained from a widevariety of known sources. For example, a variety of antibody secretinghybridoma lines are available from the American Type Culture Collection(ATCC, Manassas, Va.). A large number of antibodies against variousdisease targets, including but not limited to tumor-associated antigens,have been deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572; 856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206, 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art. Isolated antibodies may be conjugated totherapeutic agents, such as camptothecins, using the techniquesdisclosed herein.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In the XENOMOUSE® and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained, forexample, by pepsin or papain digestion of whole antibodies byconventional methods. For example, antibody fragments may be produced byenzymatic cleavage of antibodies with pepsin to provide a 5S fragmentdenoted F(ab′)₂. This fragment may be further cleaved using a thiolreducing agent and, optionally, a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab fragments and an Fc fragment.Exemplary methods for producing antibody fragments are disclosed in U.S.Pat. No. 4,036,945; U.S. Pat. No. 4,331,647; Nisonoff et al., 1960,Arch. Biochem. Biophys., 89:230; Porter, 1959, Biochem. J., 73:119;Edelman et al., 1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press),and Coligan et al. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (JohnWiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Target Antigens and Exemplary Antibodies

In a preferred embodiment, antibodies are used that recognize and/orbind to antigens that are expressed at high levels on target cells andthat are expressed predominantly or exclusively on diseased cells versusnormal tissues. More preferably, the antibodies internalize rapidlyfollowing binding. An exemplary rapidly internalizing antibody is theLL1 (anti-CD74) antibody, with a rate of internalization ofapproximately 8×10⁶ antibody molecules per cell per day (e.g., Hansen etal., 1996, Biochem J. 320:293-300). Thus, a “rapidly internalizing”antibody may be one with an internalization rate of about 1×10⁶ to about1×10⁷ antibody molecules per cell per day. Antibodies of use in theclaimed compositions and methods may include MAbs with properties asrecited above. Exemplary antibodies of use for therapy of, for example,cancer include but are not limited to LL1 (anti-CD74), LL2 or RFB4(anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20),obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor),nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7(anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 orKC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, alsoknown as CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), R1(anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomabtiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20);PAM4 (aka clivatuzumab, anti-mucin) and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20050271671;20060193865; 20060210475; 20070087001; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4,alpha-fetoprotein (AFP), VEGF (e.g., AVASTIN®, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1 (TROP-2), EGP-2 (e.g.,17-1A), EGF receptor (ErbB1) (e.g., ERBITUX®), ErbB2, ErbB3, Factor H,FHL-1, Flt-3, folate receptor, Ga 733,GRO-β, HMGB-1, hypoxia induciblefactor (HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF),IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R,IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigen to which L243binds, CD66 antigens, i.e., CD66a-d or a combination thereof, MAGE,mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration-inhibitory factor(MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor (P1GF),PSA (prostate-specific antigen), PSMA, PAM4 antigen, PD-1 receptor,NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100,tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumor necrosisantigens, tumor angiogenesis antigens, TNF-α, TRAIL receptor (R1 andR2), TROP-2, VEGFR, RANTES, T101, as well as cancer stem cell antigens,complement factors C3, C3a, C3b, C5a, C5, and an oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Perris, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab and hRFB4), CD74(milatuzumab), CD19 (hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see,e.g., Winer et al., 2011, Nature Med 17:610-18). Anti-CD3 antibodiesalso have been proposed for therapy of type 1 diabetes (Cernea et al.,2010, Diabetes Metab Rev 26:602-05).

The pharmaceutical composition of the present invention may be used totreat a subject having a metabolic disease, such amyloidosis, or aneurodegenerative disease, such as Alzheimer's disease. Bapineuzumab isin clinical trials for Alzheimer's disease therapy. Other antibodiesproposed for therapy of Alzheimer's disease include Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,and solanezumab. Infliximab, an anti-TNF-α antibody, has been reportedto reduce amyloid plaques and improve cognition.

In a preferred embodiment, diseases that may be treated using theclaimed compositions and methods include cardiovascular diseases, suchas fibrin clots, atherosclerosis, myocardial ischemia and infarction.Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and inclinical trials as imaging agents for disclosing said clots andpulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL), anti-MIF, and anti-CD74 (e.g., hLL1) antibodies canbe used to target atherosclerotic plaques. Abciximab (anti-glycoproteinIIb/IIIa) has been approved for adjuvant use for prevention ofrestenosis in percutaneous coronary interventions and the treatment ofunstable angina (Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3antibodies have been reported to reduce development and progression ofatherosclerosis (Steffens et al., 2006, Circulation 114:1977-84).Antibodies against oxidized LDL induced a regression of establishedatherosclerosis in a mouse model (Ginsberg, 2007, J Am Coll Cardiol52:2319-21). Anti-ICAM-1 antibody was shown to reduce ischemic celldamage after cerebral artery occlusion in rats (Zhang et al., 1994,Neurology 44:1747-51). Commercially available monoclonal antibodies toleukocyte antigens are represented by: OKT anti-T-cell monoclonalantibodies (available from Ortho Pharmaceutical Company) which bind tonormal T-lymphocytes; the monoclonal antibodies produced by thehybridomas having the ATCC accession numbers HB44, HB55, HB12, HB78 andHB2; G7E11, W8E7, NKP15 and GO22 (Becton Dickinson); NEN9.4 (New EnglandNuclear); and FMC11 (Sera Labs). A description of antibodies againstfibrin and platelet antigens is contained in Knight, Semin. Nucl. Med.,20:52-67 (1990).

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)). A review of the use of CD74antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007Sep. 15; 13(18 Pt 2):5556s-5563s, incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any chemotherapeutic moiety it carries. Thisallows a high, and therapeutic, concentration of LL1-chemotherapeuticdrug conjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug conjugates are cycled through lysosomes andendosomes, and the chemotherapeutic moiety is released in an active formwithin the target cells.

In another preferred embodiment, the therapeutic conjugates can be usedagainst pathogens, since antibodies against pathogens are known. Forexample, antibodies and antibody fragments which specifically bindmarkers produced by or associated with infectious lesions, includingviral, bacterial, fungal and parasitic infections, for example caused bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi, andviruses, and antigens and products associated with such microorganismshave been disclosed, inter alia, in Hansen et al., U.S. Pat. No.3,927,193 and Goldenberg U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544,4,468,457, 4,444,744, 4,818,709 and 4,624,846, the Examples section ofeach incorporated herein by reference, and in Reichert and Dewitz, citedabove. A review listing antibodies against infectious organisms(antitoxin and antiviral antibodies), as well as other targets, iscontained in Casadevall, Clin Immunol 1999; 93(1):5-15, incorporatedherein by reference.

In a preferred embodiment, the pathogens are selected from the groupconsisting of HIV virus, Mycobacterium tuberculosis, Streptococcusagalactiae, methicillin-resistant Staphylococcus aureus, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcusneoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus,cytomegalovirus, herpes simplex virus I, herpes simplex virus II, humanserum parvo-like virus, respiratory syncytial virus, varicella-zostervirus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus,human T-cell leukemia viruses, Epstein-Barr virus, murine leukemiavirus, mumps virus, vesicular stomatitis virus, sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus40, mouse mammary tumor virus, dengue virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae, as disclosed inU.S. Pat. No. 6,440,416, the Examples section of which is incorporatedherein by reference.

In a more preferred embodiment, drug conjugates of the present inventioncomprising anti-gp120 and other such anti-HIV antibodies can be used astherapeutics for HIV in AIDS patients; and drug conjugates of antibodiesto Mycobacterium tuberculosis are suitable as therapeutics fordrug-refractive tuberculosis. Fusion proteins of anti-gp120 MAb (antiHIV MAb) and a toxin, such as Pseudomonas exotoxin, have been examinedfor antiviral properties (Van Oigen et al., J Drug Target, 5:75-91,1998). Attempts at treating HIV infection in AIDS patients failed,possibly due to insufficient efficacy or unacceptable host toxicity. TheCPT drug conjugates of the present invention advantageously lack suchtoxic side effects of protein toxins, and are therefore advantageouslyused in treating HIV infection in AIDS patients. These drug conjugatescan be given alone or in combination with other antibiotics ortherapeutic agents that are effective in such patients when given alone.Candidate anti-HIV antibodies include the P4/D10 anti-envelope antibodydescribed by Johansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), aswell as the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. No. 5,831,034, U.S. Pat. No.5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos etal., Antimicrob. Agents Chemother. 2006; 50(5):1773-9, all incorporatedherein by reference. A preferred targeting agent for HIV is variouscombinations of these antibodies in order to overcome resistance.

Antibodies of use to treat autoimmune disease or immune systemdysfunctions (e.g., graft-versus-host disease, organ transplantrejection) are known in the art and may be conjugated to SN-38 using thedisclosed methods and compositions. Antibodies of use to treatautoimmune/immune dysfunction disease may bind to exemplary antigensincluding, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4,CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43,CD45, CD55, TNF-alpha, interferon and HLA-DR. Antibodies that bind tothese and other target antigens, discussed above, may be used to treatautoimmune or immune dysfunction diseases. Autoimmune diseases that maybe treated with immunoconjugates may include acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

The antibodies discussed above and other known antibodies againstdisease-associated antigens may be used as CPT-conjugates, morepreferably SN-38-conjugates, in the practice of the claimed methods andcompositions.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). A preferred bispecific antibody is an anti-CD3×anti-CD19antibody. In alternative embodiments, an anti-CD3 antibody or fragmentthereof may be attached to an antibody or fragment against anotherB-cell associated antigen, such as anti-CD3×anti-CD20,anti-CD3×anti-CD22, anti-CD3×anti-HLA-DR or anti-CD3×anti-CD74. Incertain embodiments, the techniques and compositions for therapeuticagent conjugation disclosed herein may be used with bispecific ormultispecific antibodies as the targeting moieties.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez,et al. Nature, 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of each ofwhich is incorporated herein by reference. Reduction of the peptidelinker length to less than 12 amino acid residues prevents pairing ofV_(H) and V_(L) domains on the same chain and forces pairing of V_(H)and V_(L) domains with complementary domains on other chains, resultingin the formation of functional multimers. Polypeptide chains of V_(H)and V_(L) domains that are joined with linkers between 3 and 12 aminoacid residues form predominantly dimers (termed diabodies). With linkersbetween 0 and 2 amino acid residues, trimers (termed triabody) andtetramers (termed tetrabody) are favored, but the exact patterns ofoligomerization appear to depend on the composition as well as theorientation of V-domains (V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), inaddition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of whichincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens. Some ofthe more preferred target combinations include theose listed in Table 1.This is a list of examples of preferred combinations, but is notintended to be exhaustive.

TABLE 1 Some Examples of multispecific antibodies. First target Secondtarget MIF A second proinflammatory effector cytokine, especiallyHMGB-1, TNF-α, IL-1, or IL-6 MIF Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP-1A, or MIP-1B MIF Proinflammatory effectorreceptor, especially IL-6R, IL-13R, and IL-15R MIF Coagulation factor,especially TF or thrombin MIF Complement factor, especially C3, C5, C3a,or C5a MIF Complement regulatory protein, especially CD46, CD55, CD59,and mCRP MIF Cancer associated antigen or receptor HMGB-1 A secondproinflammatory effector cytokine, especially MIF, TNF-α, IL-1, or IL-6HMGB-1 Proinflammatory effector chemokine, especially MCP-1, RANTES,MIP-1A, or MIP-1B HMGB-1 Proinflammatory effector receptor especiallyMCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1 Coagulation factor, especiallyTF or thrombin HMGB-1 Complement factor, especially C3, C5, C3a, or C5aHMGB-1 Complement regulatory protein, especially CD46, CD55, CD59, andmCRP HMGB-1 Cancer associated antigen or receptor TNF-α A secondproinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 TNF-α Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP-1A, or MIP-1B TNF-α Proinflammatory effector receptor,especially IL-6R IL-13R, and IL-15R TNF-α Coagulation factor, especiallyTF or thrombin TNF-α Complement factor, especially C3, C5, C3a, or C5aTNF-α Complement regulatory protein, especially CD46, CD55, CD59, andmCRP TNF-α Cancer associated antigen or receptor LPS Proinflammatoryeffector cytokine, especially MIF, HMGB-1, TNF-α, IL-1, or IL-6 LPSProinflammatory effector chemokine, especially MCP-1, RANTES, MIP-1A, orMIP-1B LPS Proinflammatory effector receptor, especially IL-6R IL-13R,and IL-15R LPS Coagulation factor, especially TF or thrombin LPSComplement factor, especially C3, C5, C3a, or C5a LPS Complementregulatory protein, especially CD46, CD55, CD59, and mCRP TF orProinflammatory effector cytokine, especially MIF, thrombin HMGB-1,TNF-α, IL-1, or IL-6 TF or Proinflammatory effector chemokine,especially MCP-1, thrombin RANTES, MIP-1A, or MIP-1B TF orProinflammatory effector receptor, especially IL-6R thrombin IL-13R, andIL-15R TF or Complement factor, especially C3, C5, C3a, or C5a thrombinTF or Complement regulatory protein, especially CD46, CD55, thrombinCD59, and mCRP TF or Cancer associated antigen or receptor thrombin

Still other combinations, such as are preferred for cancer therapies,include CD20+CD22 antibodies, CD74+CD20 antibodies, CD74+CD22antibodies, CEACAM5 (CEA)+CEACAM6 (NCA) antibodies, insulin-like growthfactor (ILGF)+CEACAM5 antibodies, EGP-1 (e.g., RS-7)+ILGF antibodies,CEACAM5+EGFR antibodies, IL6+CEACAM6 antibodies. Such antibodies neednot only be used in combination, but can be combined as fusion proteinsof various forms, such as IgG, Fab, scFv, and the like, as described inU.S. Pat. Nos. 6,083,477; 6,183,744 and 6,962,702 and U.S. PatentApplication Publication Nos. 20030124058; 20030219433; 20040001825;20040202666; 20040219156; 20040219203; 20040235065; 20050002945;20050014207; 20050025709; 20050079184; 20050169926; 20050175582;20050249738; 20060014245 and 20060034759, the Examples section of eachincorporated herein by reference.

DOCK-AND-LOCK™ (DNL™)

In preferred embodiments, a bivalent or multivalent antibody is formedas a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070; 7,871,622;7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398; 8,003,111 and8,034,352, the Examples section of each of which is incorporated hereinby reference.) Generally, the technique takes advantage of the specificand high-affinity binding interactions that occur between a dimerizationand docking domain (DDD) sequence of the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequencederived from any of a variety of AKAP proteins (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein,peptide or other molecule. Because the DDD sequences spontaneouslydimerize and bind to the AD sequence, the technique allows the formationof complexes between any selected molecules that may be attached to DDDor AD sequences.

Although the standard DNL™ complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL™complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL™ complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL™constructs of different stoichiometry may be produced and used (see,e.g., U.S. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL™construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

In various embodiments, an antibody or antibody fragment may beincorporated into a DNL™ complex by, for example, attaching a DDD or ADmoiety to the C-terminal end of the antibody heavy chain, as describedin detail below. In more preferred embodiments, the DDD or AD moiety,more preferably the AD moiety, may be attached to the C-terminal end ofthe antibody light chain (see, e.g., U.S. patent application Ser. No.13/901,737, filed May 24, 2013, the Examples section of which isincorporated herein by reference.)

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL™ complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RIβ(SEQ ID NO: 9) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILAPKA RIIα (SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Can et al., 2001, J Biol Chem 276:17332-38; Altoet al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al.,2006, Biochem J396:297-306; Stokka et al., 2006, Biochem J400:493-99;Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408, the entire text of each of which is incorporated herein byreference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 2 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1). Consensus  sequence disclosed as SEQ ID NO: 87.S H I Q I P P G L T E L L Q G Y T V E V L R T K N A S D N A S D K R Q QP P D L V E F A V E Y F T R L R E A R A N N E D L D S K K D L K L I I IV V V THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 12)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD 1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)

TABLE 3 Conservative Amino Acid Substitutions in AD1(SEQ ID NO: 3). Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y LA K Q I V D N A I Q Q A N L D F I R N E Q N N L V T V I S VNIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA (SEQ ID NO: 37)QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA (SEQ ID NO: 41)QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA (SEQ ID NO: 43)QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA (SEQ ID NO: 45)QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV (SEQ ID NO: 49)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL™ constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

Super AKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 50)Alternative AKAP sequences QIEYKAKQIVDHAIHQA (SEQ ID NO: 51)QIEYHAKQIVDHAIHQA (SEQ ID NO: 52) QIEYVAKQIVDHAIHQA (SEQ ID NO: 53)

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL PLEYQAGLLVQNAIQQAI (SEQ ID NO: 54) AKAP79LLIETASSLVKNAIQLSI (SEQ ID NO: 55) AKAP-Lbc LIEEAASRIVDAVIEQVK(SEQ ID NO: 56) RI-Specific AKAPs AKAPce ALYQFADRFSELVISEAL(SEQ ID NO: 57) RIAD LEQVANQLADQIIKEAT (SEQ ID NO: 58) PV38FEELAWKIAKMIWSDVF (SEQ ID NO: 59) Dual-Specificity AKAPs AKAP7ELVRLSKRLVENAVLKAV (SEQ ID NO: 60) MAP2D TAEEVSARIVQVVTAEAV(SEQ ID NO: 61) DAKAP1 QIKQAAFQLISQVILEAT (SEQ ID NO: 62) DAKAP2LAWKIAKMIVSDVMQQ (SEQ ID NO: 63)

Stokka et al. (2006, Biochem J400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 64) RIAD LEQYANQLADQIIKEATE(SEQ ID NO: 65) PV-38 FEELAWKIAKMIWSDVFQQC (SEQ ID NO: 66)

Hundsrucker et al. (2006, Biochem J396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 3) AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)

Can et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare numerous possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5 Conservative Amino Acid Substitutions in DDD1(SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 89. S H I Q IP P G L T E L L Q G Y T V E V L R T N S I L A Q Q P P D L V E F A V E YF T R L R E A R A N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:85) and veltuzumab (SEQ IDNO:86).

Rituximab heavy chain variable region sequence (SEQ ID NO: 85)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 86 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotypoe characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies. Table 6compares the allotype sequences of rituximab vs. veltuzumab. As shown inTable 6, rituximab (G1m17,1) is a DEL allotype IgG1, with an additionalsequence variation at Kabat position 214 (heavy chain CH1) of lysine inrituximab vs. arginine in veltuzumab. It has been reported thatveltuzumab is less immunogenic in subjects than rituximab (see, e.g.,Morchhauser et al., 2009, J Clin Oncol 27:3346-53; Goldenberg et al.,2009, Blood 113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), aneffect that has been attributed to the difference between humanized andchimeric antibodies. However, the difference in allotypes between theEEM and DEL allotypes likely also accounts for the lower immunogenicityof veltuzumab.

TABLE 6 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17, 1 K 17 D/L 1 A — Veltuzumab G1m3R  3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL™ constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Avimers

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220). The resultingmultidomain proteins may comprise multiple independent binding domains,that may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, DDD and/orAD sequences for use in the claimed methods and compositions. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756, 20050048512, 20050053973, 20050089932 and 20050221384, theExamples section of each of which is incorporated herein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods forpreparing a phage library. The phage display technique involvesgenetically manipulating bacteriophage so that small peptides can beexpressed on their surface (Smith and Scott, 1985, Science228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. 21:228-257). Inaddition to peptides, larger protein domains such as single-chainantibodies may also be displayed on the surface of phage particles (Arapet al., 1998, Science 279:377-380).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, theExamples section of each incorporated herein by reference. Methods forpreparation and screening of aptamers that bind to particular targets ofinterest are well known, for example U.S. Pat. No. 5,475,096 and U.S.Pat. No. 5,270,163, the Examples section of each incorporated herein byreference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

Affibodies and Fynomers

Certain alternative embodiments may utilize affibodies in place ofantibodies. Affibodies are commercially available from Affibody AB(Solna, Sweden). Affibodies are small proteins that function as antibodymimetics and are of use in binding target molecules. Affibodies weredeveloped by combinatorial engineering on an alpha helical proteinscaffold (Nord et al., 1995, Protein Eng 8:601-8; Nord et al., 1997, NatBiotechnol 15:772-77). The affibody design is based on a three helixbundle structure comprising the IgG binding domain of protein A (Nord etal., 1995; 1997). Affibodies with a wide range of binding affinities maybe produced by randomization of thirteen amino acids involved in the Fcbinding activity of the bacterial protein A (Nord et al., 1995; 1997).After randomization, the PCR amplified library was cloned into aphagemid vector for screening by phage display of the mutant proteins.The phage display library may be screened against any known antigen,using standard phage display screening techniques (e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, Quart. J. Nucl.Med. 43:159-162), in order to identify one or more affibodies againstthe target antigen.

A ¹⁷⁷Lu-labeled affibody specific for HER2/neu has been demonstrated totarget HER2-expressing xenografts in vivo (Tolmachev et al., 2007,Cancer Res 67:2773-82). Although renal toxicity due to accumulation ofthe low molecular weight radiolabeled compound was initially a problem,reversible binding to albumin reduced renal accumulation, enablingradionuclide-based therapy with labeled affibody (Id.).

The feasibility of using radiolabeled affibodies for in vivo tumorimaging has been recently demonstrated (Tolmachev et al., 2011,Bioconjugate Chem 22:894-902). A maleimide-derivatized NOTA wasconjugated to the anti-HER2 affibody and radiolabeled with ¹¹¹In (Id.).Administration to mice bearing the HER2-expressing DU-145 xenograft,followed by gamma camera imaging, allowed visualization of the xenograft(Id.).

Fynomers can also bind to target antigens with a similar affinity andspecificity to antibodies. Fynomers are based on the human Fyn SH3domain as a scaffold for assembly of binding molecules. The Fyn SH3domain is a fully human, 63 amino acid protein that can be produced inbacteria with high yields. Fynomers may be linked together to yield amultispecific binding protein with affinities for two or more differentantigen targets. Fynomers are commercially available from COVAGEN AG(Zurich, Switzerland).

The skilled artisan will realize that affibodies or fynomers may be usedas targeting molecules in the practice of the claimed methods andcompositions.

Conjugation Protocols

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that is facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering a therapeutically effective amount ofa therapeutic conjugate as described herein to a subject. Diseases thatmay be treated with the therapeutic conjugates described herein include,but are not limited to B-cell malignancies (e.g., non-Hodgkin'slymphoma, mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma,diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma,acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cellleukemia) using, for example an anti-CD22 antibody such as the hLL2 MAb(epratuzumab, see U.S. Pat. No. 6,183,744), against another CD22 epitope(hRFB4) or antibodies against other B cell antigens, such as CD19, CD20,CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD74, CD80 or HLA-DR. Otherdiseases include, but are not limited to, adenocarcinomas ofendodermally-derived digestive system epithelia, cancers such as breastcancer and non-small cell lung cancer, and other carcinomas, sarcomas,glial tumors, myeloid leukemias, etc. In particular, antibodies againstan antigen, e.g., an oncofetal antigen, produced by or associated with amalignant solid tumor or hematopoietic neoplasm, e.g., agastrointestinal, stomach, colon, esophageal, liver, lung, breast,pancreatic, liver, prostate, ovarian, testicular, brain, bone orlymphatic tumor, a sarcoma or a melanoma, are advantageously used. Suchtherapeutics can be given once or repeatedly, depending on the diseasestate and tolerability of the conjugate, and can also be used optionallyin combination with other therapeutic modalities, such as surgery,external radiation, radioimmunotherapy, immunotherapy, chemotherapy,antisense therapy, interference RNA therapy, gene therapy, and the like.Each combination will be adapted to the tumor type, stage, patientcondition and prior therapy, and other factors considered by themanaging physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize.

In a preferred embodiment, therapeutic conjugates comprising ananti-EGP-1 (anti-TROP-2) antibody such as the hRS7 MAb can be used totreat carcinomas such as carcinomas of the esophagus, pancreas, lung,stomach, colon and rectum, urinary bladder, breast, ovary, uterus,kidney and prostate, as disclosed in U.S. Pat. Nos. 7,238,785; 7,517,964and 8,084,583, the Examples section of which is incorporated herein byreference. An hRS7 antibody is a humanized antibody that comprises lightchain complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO:90); CDR2 (SASYRYT, SEQ ID NO:91); and CDR3(QQHYITPLT, SEQ ID NO:92) and heavy chain CDR sequences CDR1 (NYGMN, SEQID NO:93); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:94) and CDR3(GGFGSSYWYFDV, SEQ ID NO:95)

In another preferred embodiment, therapeutic conjugates comprising ananti-CEACAM5 antibody (e.g., hMN-14, labretuzumab) and/or ananti-CEACAM6 antibody (e.g., hMN-3 or hMN-15) may be used to treat anyof a variety of cancers that express CEACAM5 and/or CEACAM6, asdisclosed in U.S. Pat. Nos. 7,541,440; 7,951,369; 5,874,540; 6,676,924and 8,267,865, the Examples section of each incorporated herein byreference. Solid tumors that may be treated using anti-CEACAM5,anti-CEACAM6, or a combination of the two include but are not limited tobreast, lung, pancreatic, esophageal, medullary thyroid, ovarian, colon,rectum, urinary bladder, mouth and stomach cancers. A majority ofcarcinomas, including gastrointestinal, respiratory, genitourinary andbreast cancers express CEACAM5 and may be treated with the subjectimmunoconjugates. An hMN-14 antibody is a humanized antibody thatcomprises light chain variable region CDR sequences CDR1 (KASQDVGTSVA;SEQ ID NO:96), CDR2 (WTSTRHT; SEQ ID NO:97), and CDR3 (QQYSLYRS; SEQ IDNO:98), and the heavy chain variable region CDR sequences CDR1 (TYWMS;SEQ ID NO:99), CDR2 (EIHPDSSTINYAPSLKD; SEQ ID NO:100) and CDR3(LYFGFPWFAY; SEQ ID NO:101). An hMN-3 antibody is a humanized antibodythat comprises light chain variable region CDR sequences CDR1(RSSQSIVHSNGNTYLE, SEQ ID NO:102), CDR2 (KVSNRFS, SEQ ID NO:103) andCDR3 (FQGSHVPPT, SEQ ID NO:104) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:105), CDR2 (WINTYTGEPTYADDFKG, SEQ ID NO:106) and CDR3(KGWMDFNSSLDY, SEQ ID NO:107). An hMN-15 antibody is a humanizedantibody that comprises light chain variable region CDR sequencesSASSRVSYIH (SEQ ID NO:108); GTSTLAS (SEQ ID NO:109); and QQWSYNPPT (SEQID NO:110); and heavy chain variable region CDR sequences DYYMS (SEQ IDNO:111); FIANKANGHTTDYSPSVKG (SEQ ID NO:112); and DMGIRWNFDV (SEQ IDNO:113).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD74 antibody (e.g., hLL1, milatuzumab, disclosed in U.S. Pat. Nos.7,074,403; 7,312,318; 7,772,373; 7,919,087 and 7,931,903, the Examplessection of each incorporated herein by reference) may be used to treatany of a variety of cancers that express CD74, including but not limitedto renal, lung, intestinal, stomach, breast, prostate or ovarian cancer,as well as several hematological cancers, such as multiple myeloma,chronic lymphocytic leukemia, acute lymphoblastic leukemia, non-Hodgkinlymphoma, and Hodgkin lymphoma. An hLL1 antibody is a humanized antibodycomprising the light chain CDR sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ IDNO:114), CDR2 (TVSNRFS; SEQ ID NO:115), and CDR3 (SQSSHVPPT; SEQ IDNO:116) and the heavy chain variable region CDR sequences CDR1 (NYGVN;SEQ ID NO:117), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:118), and CDR3(SRGKNEAWFAY; SEQ ID NO:119).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD22 antibody (e.g., hLL2, epratuzumab, disclosed in U.S. Pat. Nos.5,789,554; 6,183,744; 6,187,287; 6,306,393; 7,074,403 and 7,641,901, theExamples section of each incorporated herein by reference, or thechimeric or humanized RFB4 antibody) may be used to treat any of avariety of cancers that express CD22, including but not limited toindolent forms of B-cell lymphomas, aggressive forms of B-celllymphomas, chronic lymphatic leukemias, acute lymphatic leukemias,non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt lymphoma, follicularlymphoma or diffuse B-cell lymphoma. An hLL2 antibody is a humanizedantibody comprising light chain CDR sequences CDR1 (KSSQSVLYSANHKYLA,SEQ ID NO:120), CDR2 (WASTRES, SEQ ID NO:121), and CDR3 (HQYLSSWTF, SEQID NO:122) and the heavy chain CDR sequences CDR1 (SYWLH, SEQ IDNO:123), CDR2 (YINPRNDYTEYNQNFKD, SEQ ID NO:124), and CDR3 (RDITTFY, SEQID NO:125)

In a preferred embodiment, therapeutic conjugates comprising anti-CSApantibodies, such as the hMu-9 MAb, can be used to treat colorectal, aswell as pancreatic and ovarian cancers as disclosed in U.S. Pat. Nos.6,962,702; 7,387,772; 7,414,121; 7,553,953; 7,641,891 and 7,670,804, theExamples section of each incorporated herein by reference. In addition,therapeutic conjugates comprising the hPAM4 MAb can be used to treatpancreatic cancer or other solid tumors, as disclosed in U.S. Pat. Nos.7,238,786 and 7,282,567, the Examples section of each incorporatedherein by reference. An hMu-9 antibody is a humanized antibodycomprising light chain CDR sequences CDR1 (RSSQSIVHSNGNTYLE, SEQ IDNO:126), CDR2 (KVSNRFS, SEQ ID NO:127), and CDR3 (FQGSRVPYT, SEQ IDNO:128), and heavy chain variable CDR sequences CDR1 (EYVIT, SEQ IDNO:129), CDR2 (EIYPGSGSTSYNEKFK, SEQ ID NO:130), and CDR3 (EDL, SEQ IDNO:131). An hPAM4 antibody is a humanized antibody comprising lightchain variable region CDR sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:132);CDR2 (STSNLAS, SEQ ID NO:133); and CDR3 (HQWNRYPYT, SEQ ID NO:134); andheavy chain CDR sequences CDR1 (SYVLH, SEQ ID NO:135); CDR2(YINPYNDGTQYNEKFKG, SEQ ID NO:136) and CDR3 (GFGGSYGFAY, SEQ ID NO:137).

In another preferred embodiment, therapeutic conjugates comprising ananti-AFP MAb, such as IMMU31, can be used to treat hepatocellularcarcinoma, germ cell tumors, and other AFP-producing tumors usinghumanized, chimeric and human antibody forms, as disclosed in U.S. Pat.No. 7,300,655, the Examples section of which is incorporated herein byreference. An IMMU31 antibody is a humanized antibody comprising theheavy chain CDR sequences CDR1 (SYVIH, SEQ ID NO:138), CDR2(YIHPYNGGTKYNEKFKG, SEQ ID NO:139) and CDR3 (SGGGDPFAY, SEQ ID NO:140)and the light chain CDR1 (KASQDINKYIG, SEQ ID NO:141), CDR2 (YTSALLP,SEQ ID NO:142) and CDR3 (LQYDDLWT, SEQ ID NO:143).

In another preferred embodiment, therapeutic conjugates comprising ananti-HLA-DR MAb, such as hL243, can be used to treat lymphoma, leukemia,cancers of the skin, esophagus, stomach, colon, rectum, pancreas, lung,breast, ovary, bladder, endometrium, cervix, testes, kidney, liver,melanoma or other HLA-DR-producing tumors, as disclosed in U.S. Pat. No.7,612,180, the Examples section of which is incorporated herein byreference. An hL243 antibody is a humanized antibody comprising theheavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:144), CDR2(WINTYTREPTYADDFKG, SEQ ID NO:145), and CDR3 (DITAVVPTGFDY, SEQ IDNO:146) and light chain CDR sequences CDR1 (RASENIYSNLA, SEQ ID NO:147),CDR2 (AASNLAD, SEQ ID NO:148), and CDR3 (QHFWTTPWA, SEQ ID NO:149).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD20 MAb, such as veltuzumab (hA20), 1F5, obinutuzumab (GA101), orrituximab, can be used to treat lymphoma, leukemia, immunethrombocytopenic purpura, systemic lupus erythematosus, Sjögren'ssyndrome, Evans syndrome, arthritis, arteritis, pemphigus vulgaris,renal graft rejection, cardiac graft rejection, rheumatoid arthritis,Burkitt lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, smalllymphocytic lymphoma, diffuse B-cell lymphoma, marginal zone lymphoma,chronic lymphocytic leukemia, acute lymphocytic leukemia, Type Idiabetes mellitus, GVHD, multiple sclerosis or multiple myeloma, asdisclosed in U.S. Pat. Nos. 7,435,803 or 8,287,864, the Examples sectionof each incorporated herein by reference. An hA20 (veltuzumab) antibodyis a humanized antibody comprising the light chain CDR sequences CDRL1(RASSSVSYIH, SEQ ID NO:150), CDRL2 (ATSNLAS, SEQ ID NO:151) and CDRL3(QQWTSNPPT, SEQ ID NO:152) and heavy chain CDR sequences CDRH1 (SYNMH,SEQ ID NO:153), CDRH2 (AIYPGNGDTSYNQKFKG, SEQ ID NO:154) and CDRH3(STYYGGDWYFDV, SEQ ID NO:155).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD19 MAb, such as hA19, can be used to treat B-cell relatedlymphomas and leukemias, such as non-Hodgkin's lymphoma, chroniclymphocytic leukemia or acute lymphoblastic leukemia. Other diseasestates that may be treated include autoimmune diseases, such as acute orchronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosurn, Takayasu's arteritis, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, Sjögren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosingalveolitis, as disclosed in U.S. Pat. Nos. 7,109,304, 7,462,352,7,902,338, 8,147,831 and 8,337,840, the Examples section of eachincorporated herein by reference. An hA19 antibody is a humanizedantibody comprising the light chain CDR sequences CDR1 KASQSVDYDGDSYLN(SEQ ID NO: 156); CDR2 DASNLVS (SEQ ID NO: 157); and CDR3 QQSTEDPWT (SEQID NO: 158) and the heavy chain CDR sequences CDR1 SYWMN (SEQ ID NO:159); CDR2 QIWPGDGDTNYNGKFKG (SEQ ID NO: 160) and CDR3 RETTTVGRYYYAMDY(SEQ ID NO: 161).

In another preferred embodiment, therapeutic conjugates comprisinganti-tenascin antibodies can be used to treat hematopoietic and solidtumors, and conjugates comprising antibodies to tenascin can be used totreat solid tumors, preferably brain cancers like glioblastomas.

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy.

In another preferred embodiment, the therapeutic conjugates can be usedagainst pathogens, since antibodies against pathogens are known. Forexample, antibodies and antibody fragments which specifically bindmarkers produced by or associated with infectious lesions, includingviral, bacterial, fungal and parasitic infections, for example caused bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi, andviruses, and antigens and products associated with such microorganismshave been disclosed, inter alia, in Hansen et al., U.S. Pat. No.3,927,193 and Goldenberg U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544,4,468,457, 4,444,744, 4,818,709 and 4,624,846, the Examples section ofeach incorporated herein by reference, and in Reichert and Dewitz, citedabove. In a preferred embodiment, the pathogens are selected from thegroup consisting of HIV virus, Mycobacterium tuberculosis, Streptococcusagalactiae, methicillin-resistant Staphylococcus aureus, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcusneoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus,cytomegalovirus, herpes simplex virus I, herpes simplex virus II, humanserum parvo-like virus, respiratory syncytial virus, varicella-zostervirus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus,human T-cell leukemia viruses, Epstein-Barr virus, murine leukemiavirus, mumps virus, vesicular stomatitis virus, sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus40, mouse mammary tumor virus, dengue virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae, as disclosed inU.S. Pat. No. 6,440,416, the Examples section of which is incorporatedherein by reference.

In a more preferred embodiment, drug conjugates of the present inventioncomprising anti-gp120 and other such anti-HIV antibodies can be used astherapeutics for HIV in AIDS patients; and drug conjugates of antibodiesto Mycobacterium tuberculosis are suitable as therapeutics fordrug-refractive tuberculosis. Fusion proteins of anti-gp120 MAb (antiHIV MAb) and a toxin, such as Pseudomonas exotoxin, have been examinedfor antiviral properties (Van Oigen et al., J Drug Target, 5:75-91,1998). Attempts at treating HIV infection in AIDS patients failed,possibly due to insufficient efficacy or unacceptable host toxicity. Thedrug conjugates of the present invention advantageously lack such toxicside effects of protein toxins, and are therefore advantageously used intreating HIV infection in AIDS patients. These drug conjugates can begiven alone or in combination with other antibiotics or therapeuticagents that are effective in such patients when given alone. Candidateanti-HIV antibodies include the P4/D10 anti-envelope antibody describedby Johansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), as well as theanti-HIV antibodies described and sold by Polymun (Vienna, Austria),also described in U.S. Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agents Chemother. 2006; 50(5):1773-9, all incorporated herein byreference. A preferred targeting agent for HIV is various combinationsof these antibodies in order to overcome resistance.

In a preferred embodiment, a more effective incorporation into cells andpathogens can be accomplished by using multivalent, multispecific ormultivalent, monospecific antibodies. Examples of such bivalent andbispecific antibodies are found in U.S. Pat. Nos. 7,387,772; 7,300,655;7,238,785; and 7,282,567, the Examples section of each of which isincorporated herein by reference. These multivalent or multispecificantibodies are particularly preferred in the targeting of cancers andinfectious organisms (pathogens), which express multiple antigen targetsand even multiple epitopes of the same antigen target, but which oftenevade antibody targeting and sufficient binding for immunotherapybecause of insufficient expression or availability of a single antigentarget on the cell or pathogen. By targeting multiple antigens orepitopes, said antibodies show a higher binding and residence time onthe target, thus affording a higher saturation with the drug beingtargeted in this invention.

In another preferred embodiment, the therapeutic conjugates can be usedto treat autoimmune disease or immune system dysfunction (e.g.,graft-versus-host disease, organ transplant rejection). Antibodies ofuse to treat autoimmune/immune dysfunction disease may bind to exemplaryantigens including, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2,CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16,CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L,CD41a, CD43, CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD74, CD79a,CD79b, CD117, CD138, FMC-7 and HLA-DR. Antibodies that bind to these andother target antigens, discussed above, may be used to treat autoimmuneor immune dysfunction diseases. Autoimmune diseases that may be treatedwith immunoconjugates may include acute idiopathic thrombocytopenicpurpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In another preferred embodiment, a therapeutic agent used in combinationwith the camptothecin conjugate of this invention may comprise one ormore isotopes. Radioactive isotopes useful for treating diseased tissueinclude, but are not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²²⁷Th and ²¹¹Pb. The therapeutic radionuclide preferably has adecay-energy in the range of 20 to 6,000 keV, preferably in the ranges60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,and 4,000-6,000 keV for an alpha emitter. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Th-227 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or conjugate. Macrocyclicchelates such as NOTA, DOTA, and TETA are of use with a variety ofmetals and radiometals, most particularly with radionuclides of gallium,yttrium and copper, respectively. Such metal-chelate complexes can bemade very stable by tailoring the ring size to the metal of interest.Other ring-type chelates, such as macrocyclic polyethers for complexing²²³Ra, may be used.

Therapeutic agents of use in combination with the camptothecinconjugates described herein also include, for example, chemotherapeuticdrugs such as vinca alkaloids, anthracyclines, epidophyllotoxins,taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,other camptothecins, and others from these and other classes ofanticancer agents, and the like. Other cancer chemotherapeutic drugsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839. Such agents may bepart of the conjugates described herein or may alternatively beadministered in combination with the described conjugates, either priorto, simultaneously with or after the conjugate. Alternatively, one ormore therapeutic naked antibodies as are known in the art may be used incombination with the described conjugates. Exemplary therapeutic nakedantibodies are described above.

Therapeutic agents that may be used in concert with the camptothecinconjugates also may comprise toxins conjugated to targeting moieties.Toxins that may be used in this regard include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641,and Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-43.) Additional toxins suitable for use herein are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β, -γ or -λ, and stem cell growth factor, suchas that designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

The person of ordinary skill will realize that the subjectimmunoconjugates, comprising a camptothecin conjugated to an antibody orantibody fragment, may be used alone or in combination with one or moreother therapeutic agents, such as a second antibody, second antibodyfragment, second immunoconjugate, radionuclide, toxin, drug,chemotherapeutic agent, radiation therapy, chemokine, cytokine,immunomodulator, enzyme, hormone, oligonucleotide, RNAi or siRNA. Suchadditional therapeutic agents may be administered separately, incombination with, or attached to the subject antibody-drugimmunoconjugates.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, subcutaneous, rectal, transmucosal,intestinal administration, intramuscular, intramedullary, intrathecal,direct intraventricular, intravenous, intravitreal, intraperitoneal,intranasal, or intraocular injections. The preferred routes ofadministration are parenteral. Alternatively, one may administer thecompound in a local rather than systemic manner, for example, viainjection of the compound directly into a solid tumor.

Immunoconjugates can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

In a preferred embodiment, the immunoconjugate is formulated in Good'sbiological buffer (pH 6-7), using a buffer selected from the groupconsisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (IVIES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are IVIES or MOPS, preferably in the concentration range of 20to 100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The immunoconjugate can be formulated for intravenous administrationvia, for example, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate from such a matrix depends upon the molecular weightof the immunoconjugate, the amount of immunoconjugate within the matrix,and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

Generally, the dosage of an administered immunoconjugate for humans willvary depending upon such factors as the patient's age, weight, height,sex, general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of immunoconjugate thatis in the range of from about 1 mg/kg to 24 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. A dosage of 1-20 mg/kg for a 70kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-mpatient. The dosage may be repeated as needed, for example, once perweek for 4-10 weeks, once per week for 8 weeks, or once per week for 4weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy. Preferred dosages may include, but are notlimited to, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22mg/kg and 24 mg/kg. Any amount in the range of 1 to 24 mg/kg may beused. The dosage is preferably administered multiple times, once ortwice a week. A minimum dosage schedule of 4 weeks, more preferably 8weeks, more preferably 16 weeks or longer may be used. The schedule ofadministration may comprise administration once or twice a week, on acycle selected from the group consisting of: (i) weekly; (ii) everyother week; (iii) one week of therapy followed by two, three or fourweeks off; (iv) two weeks of therapy followed by one, two, three or fourweeks off; (v) three weeks of therapy followed by one, two, three, fouror five week off; (vi) four weeks of therapy followed by one, two,three, four or five week off; (vii) five weeks of therapy followed byone, two, three, four or five week off; and (viii) monthly. The cyclemay be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.

Alternatively, an immunoconjugate may be administered as one dosageevery 2 or 3 weeks, repeated for a total of at least 3 dosages. Or,twice per week for 4-6 weeks. If the dosage is lowered to approximately200-300 mg/m² (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a70 kg patient), it may be administered once or even twice weekly for 4to 10 weeks. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months. It has been determined, however, thateven higher doses, such as 12 mg/kg once weekly or once every 2-3 weekscan be administered by slow i.v. infusion, for repeated dosing cycles.The dosing schedule can optionally be repeated at other intervals anddosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

In preferred embodiments, the immunoconjugates are of use for therapy ofcancer. Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, gastric or stomach cancer including gastrointestinalcancer, pancreatic cancer, glioblastoma multiforme, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, neuroendocrine tumors, medullary thyroid cancer,differentiated thyroid carcinoma, breast cancer, ovarian cancer, coloncancer, rectal cancer, endometrial cancer or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer,anal carcinoma, penile carcinoma, as well as head-and-neck cancer. Theterm “cancer” includes primary malignant cells or tumors (e.g., thosewhose cells have not migrated to sites in the subject's body other thanthe site of the original malignancy or tumor) and secondary malignantcells or tumors (e.g., those arising from metastasis, the migration ofmalignant cells or tumor cells to secondary sites that are differentfrom the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with immunoconjugates mayinclude acute and chronic immune thrombocytopenias, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one conjugated antibody or other targeting moiety as describedherein. If the composition containing components for administration isnot formulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

Examples

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

General

Abbreviations used below are: DCC, dicyclohexylcarbodiimide; NHS,N-hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; EEDQ,2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; MMT, monomethoxytrityl;PABOH, p-aminobenzyl alcohol; PEG, polyethylene glycol; SMCC,succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; TBAF,tetrabutylammonium fluoride; TBDMS, tert-butyldimethylsilyl chloride.

Chloroformates of hydroxy compounds in the following examples wereprepared using triphosgene and DMAP according to the procedure describedin Moon et al. (J. Medicinal Chem. 51:6916-6926, 2008). Extractivework-up refers to extraction with chloroform, dichloromethane or ethylacetate, and washing optionally with saturated bicarbonate, water, andwith saturated sodium chloride. Flash chromatography was done using230-400 mesh silica gel and methanol-dichloromethane gradient, using upto 15% v/v methanol-dichloromethane, unless otherwise stated. Reversephase HPLC was performed by Method A using a 7.8×300 mm C18 HPLC column,fitted with a precolumn filter, and using a solvent gradient of 100%solvent A to 100% solvent B in 10 minutes at a flow rate of 3 mL perminute and maintaining at 100% solvent B at a flow rate of 4.5 mL perminute for 5 or 10 minutes; or by Method B using a 4.6×30 mm XbridgeC18, 2.5 μm, column, fitted with a precolumn filter, using the solventgradient of 100% solvent A to 100% of solvent B at a flow rate of 1.5 mLper minutes for 4 min and 100% of solvent B at a flow rate of 2 mL perminutes for 1 minutes. Solvent A was 0.3% aqueous ammonium acetate, pH4.46 while solvent B was 9:1 acetonitrile-aqueous ammonium acetate(0.3%), pH 4.46. HPLC was monitored by a dual in-line absorbancedetector set at 360 nm and 254 nm.

Example 1 Preparation of CL6-SN-38

CL6-SN-38 is represented in Scheme-1. Commercially availableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N₃’; 227 mg) was activated with DCC (100 mg), NHS (56 mg), and acatalytic amount of DMAP in 10 mL of dichloromethane for 10 min. To thismixture was added L-valinol (46.3 mg), and the reaction mixture wasstirred for 1 h at ambient temperature. Filtration, followed by solventremoval and flash chromatography yielded 214 mg of clear oily material.This intermediate (160 mg) was reacted with10-O—BOC—SN-38-20-O-chloroformate, the latter generated from10-O—BOC—SN-38 (123 mg) using triphosgene and DMAP. The couplingreaction was done in 4 mL of dichloromethane for 10 min, and thereaction mixture was purified by flash chromatography to obtain 130 mg(45% yield) of product as foamy material. HPLC: t_(R) 11.80 min;electrospray mass spectrum: M+Na: m/z 1181.

The maleimide-containing acetylenic reagent, namely4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide, requiredfor click cycloaddition, was prepared by reacting 0.107 g of SMCC and0.021 mL of propargylamine (0.018 g; 1.01 equiv.) in dichloromethaneusing 1.1 equiv. of diisopropylethylamine. After 1 h, the solvent wasremoved and the product was purified by flash chromatography to obtain83 mg of the product (colorless powder). Electrospray mass spectrumshowed peaks at m/e 275 (M+H) and a base peak at m/e 192 in the positiveion mode, consistent with the structure calculated for C₁₅H₁₈N₂O₃:275.1390 (M+H). found: 275.1394 (exact mass).

The azido intermediate (126 mg) was dissolved in DMSO (1.5 mL) and water(0.4 mL), and reacted with 60 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide and 15 mgof cuprous bromide and stirred for 30 min at ambient temperature. Flashchromatography, after work up of the reaction mixture, furnished 116 mg(75% yield) of the cycloaddition product. HPLC: t_(R) 11.20 min;electrospray mass spectrum: M+H and M+Na at m/z 1433 and 1456,respectively. Finally, deprotection with a mixture of TFA (5 mL),dichloromethane (1 mL), anisole (0.1 mL) and water (0.05 mL), followedby precipitation with ether and subsequent flash chromatography yieldedthe product, CL6-SN-38, as a gummy material. HPLC: t_(R) 9.98 min;electrospray mass spectrum: M+H and M−H (negative ion mode) at m/z 1333and 1356, respectively.

Example 2 Preparation of CL7-SN-38

The synthesis is schematically shown in Scheme-2. L-Valinol (40 mg) wasreacted with commercially available Fmoc-Lys(MMT)-OH (253 mg) and EEDQ(107 mg) in 10 mL of anhydrous dichloromethane at ambient temperature,under argon, for 3 h. Extractive work up followed by flashchromatography furnished the product Fmoc-Lys(MMT)-valinol as a paleyellow liquid (200 mg; ˜70% yield). HPLC: t_(R)14.38 min; electrospraymass spectrum: M+H: m/z 727. This intermediate (200 mg) was deprotectedwith diethylamine (10 mL), and the product (135 mg) was obtained in ˜90%purity after flash chromatography. HPLC: t_(R) 10.91 min; electrospraymass spectrum: M+Na at m/z 527. This product (135 mg) was coupled withthe commercially availableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N₃’; 150 mg, 1.1 equiv.) in presence of EEDQ (72 mg, 1.1 equiv.)in 10 mL of dichloromethane, and stirred overnight at ambienttemperature. The crude material was purified by flash chromatography toobtain 240 mg of the purified product as a light yellow oil (˜87%yield). HPLC: t_(R) 11.55 min; electrospray mass spectrum: M+H and M+Naat m/z 1041 and 1063, respectively.

This intermediate (240 mg) was reacted with10-O-TBDMS-SN-38-20-O-chloroformate, the latter generated from10-O-TBDMS-SN-38 (122 mg) using triphosgene and DMAP. The couplingreaction was done in 5 mL of dichloromethane for 10 min, and thereaction mixture was purified by flash chromatography to obtain 327 mgof product as pale yellow foam. Electrospray mass spectrum: M+H at m/z1574. The entire product was reacted with 0.25 mmol of TBAF in 10 mL ofdichloromethane for 5 min, and the reaction mixture was diluted to 100mL and washed with brine.

Crude product (250 mg) was dissolved in DMSO (2 mL) and water (0.4 mL),and reacted with 114 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) and 30 mg of cuprous bromide and stirred for1 h at ambient temperature. Flash chromatography furnished 150 mg of thepenultimate intermediate. Finally, deprotection of the MMT group with amixture of TFA (0.5 mL) and anisole (0.05 mL) in dichloromethane (5 mL)for 3 min, followed by purification by flash chromatography yielded 69mg of CL7-SN-38 as a gummy material. HPLC: t_(R) 9.60 min; electrospraymass spectrum: M+H and M−H (negative ion mode) at m/z 1461 and 1459,respectively.

Example 3 Preparation of CL6-SN-38-10-O—CO₂Et

The CL6-SN-38 of Example 1 (55.4 mg) was dissolved in dichloromethane (5mL), and reacted with ethylchloroformate (13.1 mg; 11.5 μL) anddiisopropylethylamine (52.5 mg; 71 pt), and stirred for 20 min underargon. The reaction mixture was diluted with 100 mL of dichloromethane,and washed with 100 mL each of 0.1 M HCl, half saturated sodiumbicarbonate and brine, and dried. Flash chromatography, after solventremoval, furnished 59 mg of the title product. HPLC: t_(R) 10.74 min;exact mass: calc. 1404.6457 (M+H) and 1426.6276 (M+Na). found: 1404.6464(M+H) and 1426.6288 (M+Na).

Example 4 Preparation of CL7-SN-38-10-O—CO₂Et

The precursor of CL7-SN-38 of Example 2 (80 mg) was converted to the10-O-chloroformate using the procedure and purification as described inExample 3. Yield: 60 mg. HPLC: t_(R) 12.32 min; electrospray massspectrum: M+H and M−H (negative ion mode) at m/z 1806 and 1804,respectively. Deprotection of this material using dichloroacetic acidand anisole in dichloromethane gave the title product. HPLC: t_(R) 10.37min; electrospray mass spectrum: M+H at m/z 1534.

Example 5 Preparations of CL6-SN-38-10-O—COR and CL7-SN-38-10-O—COR

This Example shows that the 10-OH group of SN-38 is protected as acarbonate or an ester, instead of as ‘BOC’, such that the final productis ready for conjugation to antibodies without need for deprotecting the10-OH protecting group. This group is readily deprotected underphysiological pH conditions after in vivo administration of the proteinconjugate. In these conjugates, ‘R’ can be a substituted alkyl such as(CH₂)_(n)—N(CH₃)₂ where n is 2-10, or a simple alkyl such as (CH₂)n-CH₃where n is 0-10, or it can be an alkoxy moiety such as “CH₃—(CH₂)n-O—”where n is 0-10, or a substituted alkoxy moiety such as such asO—(CH₂)_(n)—N(CH₃)₂ where n is 2-10 and wherein the terminal amino groupis optionally in the form of a quaternary salt for enhanced aqueoussolubility, or “R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl ormethyl and n is an integer with values of 0-10. In the simplest versionof the latter category, R═“—O—(CH₂)₂—OCH₃”. These 10-hydroxy derivativesare readily prepared by treatment with the chloroformate of the chosenreagent, if the final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused. Such derivatizations are conveniently accomplished using advancedintermediates as illustrated for simple ethyl carbonates of Examples 3and 4.

Example 6 Preparation of CL2A-SN-38

To the mixture of commercially available Fmoc-Lys(MMT)-OH (0.943 g),p-aminobenzyl alcohol (0.190 g) in methylene choloride (10 mL) was addedEEDQ (0.382 g) at room temperature and stirred for 4 h. Extractive workup followed by flash chromatograph yielded 1.051 g of material as whitefoam. All HPLC analyses were performed by Method B as stated in‘General’ in section 0148. HPLC ret. time: 3.53 min., Electrospray massspectrum showed peaks at m/e 745.8 (M+H) and m/e 780.3 (M+Cl⁻),consistent with structure. This intermediate (0.93 g) was dissolved indiethylamine (10 mL) and stirred for 2 h. After solvent removal, theresidue was washed in hexane to obtain 0.6 g of the intermediate ((2) inScheme-3) as colorless precipitate (91.6% pure by HPLC). HPLC ret. time:2.06 min. Electrospray mass spectrum showed peaks at m/e 523.8 (M+H),m/e 546.2 (M+Na) and m/e 522.5 (M−H).

This crude intermediate (0.565 g) was coupled with commerciallyavailableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(′PEG-N3′, 0.627 g) using EEDQ in methylene chloride (10 mL). Solventremoval and flash chromatography yielded 0.99 g of the product ((3) inScheme-3; light yellow oil; 87% yield). HPLC ret. time: 2.45 min.Electrospray mass spectrum showed peaks at m/e 1061.3 (M+H), m/e 1082.7(M+Na) and m/e 1058.8 (M−H), consistent with structure. Thisintermediate (0.92 g) was reacted with10-O-TBDMS-SN-38-20-O-chloroformate ((5) in Scheme-3) in methylenechloride (10 mL) for 10 min under argon. The mixture was purified byflash chromatography to obtain 0.944 g as light yellow oil ((6) inScheme-3; yield=68%). HPLC ret. time: 4.18 min. To this intermediate(0.94 g) in methylene chloride (10 mL) was added the mixture of TBAF (1Min THF, 0.885 mL) and acetic acid (0.085 mL) in methylene chloride (3mL), then stirred for 10 min. The mixture was diluted with methylenechloride (100 mL), washed with 0.25 M sodium citrate and brine. Thesolvent removal yielded 0.835 g of yellow oily product. HPLC ret. time:2.80 min., (99% purity). Electrospray mass spectrum showed peaks at m/e1478 (M+H), m/e 1500.6 (M+Na), m/e 1476.5 (M−H), m/e 1590.5 (M+TFA),consistent with structure.

This azido-derivatized SN-38 intermediate (0.803 g) was reacted with4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (0.233 g)in methylene chloride (10 mL) in presence of CuBr (0.0083 g), DIEA (0.01mL) and triphenylphosphine (0.015 g), for 18 h. Extractive work up,including washing with and 0.1M EDTA (10 mL), and flash chromatographyyielded 0.891 g as yellow foam. (yield=93%), HPLC ret. time: 2.60 min.Electrospray mass spectrum showed peaks at m/e 1753.3 (M+H), m/e 1751.6(M−H), 1864.5 (M+TFA), consistent with structure. Finally, deprotectionof the penultimate intermediate (0.22 g) with a mixture ofdichloroacetic acid (0.3 mL) and anisole (0.03 mL) in methylene chloride(3 mL), followed by precipitation with ether yielded 0.18 g (97% yield)of CL2A-SN-38; (7) in Scheme-3) as light yellow powder. HPLC ret. time:1.88 min. Electrospray mass spectrum showed peaks at m/e 1480.7 (M+H),1478.5 (M−H), consistent with structure.

Example 7 Preparation of CL2E-SN-38

N,N′-dimethylethylenediamine (3 mL) in methylene chloride (50 mL) wasreacted with monomethoxytrityl chloride (1.7 g). After 1 h of stirring,the solvent was removed under reduced pressure, and the crude productwas recovered by extractive work up (yellow oil; 2.13 g). All HPLCanalyses were performed by Method B as stated in ‘General’ in section0148. HPLC ret. time: 2.28 min. This intermediate ((1) in Scheme-4; 0.93g) was added in situ to activated SN-38, and the latter ((2) inScheme-4) was prepared by reacting SN-38 (0.3 g) withp-nitrophenylchloroformate (0.185 g) and DIEA (0.293 mL) in DMF for 1 h.After removing solvent, the residue was purified on deactivated silicagel to obtain 0.442 g as white solid.

This intermediate (0.442 g) was deprotected with a mixture oftrifluoroacetic acid (1 mL) and anisole (0.1 mL) in methylene chloride(5 mL), followed by precipitation with ether to obtain 0.197 g of theproduct ((3) in Scheme-4) as white solid. This intermediate ((3); 0.197g) was coupled with activated azide-containing-dipeptideincorporated-PEG-linker ((5) in Scheme-4), which activation was done byreacting PEG-linker ((4) in Scheme-4; 0.203 g) with bis(4-nitrophenyl)carbonate (0.153 g) and DIEA (0.044 mL) in methylene chloride (8 mL).Flash chromatography yielded 0.2 g of azide-derivatized SN-38intermediate product ((6) in Scheme-4) as glassy solid. HPLC ret. time:2.8 min. Electrospray mass spectrum showed peaks at m/e 1740.5 (M+H),m/e 1762.9 (M+Na), m/e 1774.9 (M+Cl⁻), consistent with structure. Thisintermediate ((6) in Scheme-4; 0.2 g) was subjected to clickcycloaddition with4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (0.067 g)in methylene chloride in presence of CuBr (0.007 g), DIEA (0.008 mL) andtriphenylphosphine (0.012 g) for 18 h. Work up of reaction mixture,which included treatment with 0.1M EDTA, followed by flashchromatography yielded 0.08 g of the penultimate intermediate as lightyellow foam. HPLC: t_(R)=2.63 min. Electrospray mass spectrum showedpeaks at m/e 2035.9 (M+Na⁺), m/e 2047.9 (M+Cl⁻), consistent withstructure. Finally, deprotection of this intermediate (0.08 g) with amixture of trifluoroacetic acid (0.2 mL), anisole (0.12 mL) and water(0.06 mL) in methylene chloride (2 mL), followed by precipitation withether yielded 0.051 g of product, CL17-SN-38 (also referred to asCL2E-SN-38), as light yellow powder (yield=69%). HPLC ret. time: 1.95min., ˜99% purity. Electrospray mass spectrum showed peaks at m/e 1741.1(M+H), 1775.5 (M+Cl⁻), consistent with structure.

Example 8 Conjugation of Bifunctional SN-38 Products to Mildly ReducedAntibodies

The anti-CEACAM5 humanized MAb, hMN-14 (also known as labetuzumab), theanti-CD22 humanized MAb, hLL2 (also known as epratuzumab), the anti-CD20humanized MAb, hA20 (also known as veltuzumab), the anti-EGP-1 humanizedMAb, hRS7, and anti-mucin humanized MAb, hPAM4 (also known asclivatuzumab), were used in these studies. Each antibody was reducedwith dithiothreitol (DTT), used in a 50-to-70-fold molar excess, in 40mM PBS, pH 7.4, containing 5.4 mM EDTA, at 37° C. (bath) for 45 min. Thereduced product was purified by size-exclusion chromatography and/ordiafiltration, and was buffer-exchanged into a suitable buffer at pH6.5. The thiol content was determined by Ellman's assay, and was in the6.5-to-8.5 SH/IgG range. Alternatively, the antibodies were reduced withTris(2-carboxyethyl)phosphine (TCEP) in phosphate buffer at pH in therange of 5-7, followed by in situ conjugation. The reduced MAb wasreacted with ˜10-to-15-fold molar excess of ‘CL6-SN-38’ of Example 1, or‘CL7-SN-38’ of Example 2, or ‘CL6-SN-38-10-O—CO₂Et’ of Example 3, or‘CL7-SN-38-10-O—CO₂Et’ of Example 4, CL2A-SN-38 of Example 6, orCL2E-SN-38 of Example 7 using DMSO at 7-15% v/v as co-solvent, andincubating for 20 min at ambient temperature. The conjugate was purifiedby centrifuged SEC, passage through a hydrophobic column, and finally byultrafiltration-diafiltration. The product was assayed for SN-38 byabsorbance at 366 nm and correlating with standard values, while theprotein concentration was deduced from absorbance at 280 nm, correctedfor spillover of SN-38 absorbance at this wavelength. This way, theSN-38/MAb substitution ratios were determined. The purified conjugateswere stored as lyophilized formulations in glass vials, capped undervacuum and stored in a −20° C. freezer. SN-38 molar substitution ratios(MSR) obtained for some of these conjugates, which were typically in the5-to-7 range, are shown in Table 7.

TABLE 7 SN-38/MAb Molar substitution ratios (MSR) in some conjugates MAbConjugate MSR hMN-14 hMN-14-[CL2A-SN-38], using drug-linker of 6.1Example 10 hMN-14-[CL6-SN-38], using drug-linker of Example 1 6.8hMN-14-[CL7-SN-38], using drug-linker of Example 2 5.9hMN-14-[CL7-SN-38-10-O—CO₂Et], using drug- 5.8 linker of Example 4hMN-14-[CL2E-SN-38], using drug-linker of 5.9 Example 11 hRS7hRS7-CL2A-SN-38 using drug-linker of Example 10 5.8 hRS7-CL7-SN-38 usingdrug-linker of Example 2 5.9 hRS7-CL7-SN-38 (Et) using drug-linker ofExample 4 6.1 hA20 hA20-CL2A-SN-38 using drug-linker of Example 10 5.8hLL2 hLL2-CL2A-SN-38 using drug-linker of Example 10 5.7 hPAM4hPAM4-CL2A-SN-38 using drug-linker of Example 10 5.9

Example 9 In Vivo Therapeutic Efficacies in Preclinical Models of HumanPancreatic or Colon Carcinoma

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. FIG. 1 shows a Capan 1 pancreatic tumor model, whereinspecific CL2A-SN-38 conjugates of hRS7 (anti-EGP-1), hPAM4 (anti-mucin),and hMN-14 (anti-CEACAM5) antibodies showed better efficacies thancontrol hA20-CL2A-SN-38 conjugate (anti-CD20) and untreated control.Similarly in a BXPC3 model of human pancreatic cancer, the specifichRS7-CL2A-SN-38 showed better therapeutic efficacy than controltreatments (FIG. 2). Likewise, in an aggressive LS174T model of humancolon carcinoma, treatment with specific hMN-14-CL2A-SN-38 was moreefficacious than non-treatment (FIG. 3).

Example 10 In Vivo Therapy of Lung Metastases of GW-39 Human ColonicTumors in Nude Mice Using hMN-14-[CL1-SN-38] and hMN-14-[CL2-SN-38]

A lung metastatic model of colonic carcinoma was established in nudemice by i.v. injection of GW-39 human colonic tumor suspension, andtherapy was initiated 14 days later. Specific anti-CEACAM5 antibodyconjugates, hMN14-CL1-SN-38 and hMN14-CL2-SN-38, as well as nontargetinganti-CD22 MAb control conjugates, hLL2-CL1-SN-38 and hLL2-CL2-SN-38 andequidose mixtures of hMN14 and SN-38 were injected at a dose schedule ofq4dx8, using different doses. FIG. 4 (MSR=SN-38/antibody molarsubstitution ratio) shows selective therapeutic effects due to hMN-14conjugates. At equivalent dosages of 250 μg, the mice treated withhMN14-CL1-SN-38 or hMN14-CL2-SN-38 showed a median survival of greaterthan 107 days. Mice treated with the control conjugated antibodieshLL2-CL1-SN-38 and hLL2-CL2-SN-38, which do not specifically target lungcancer cells, showed median survival of 56 and 77 days, while micetreated with unconjugated hMN14 IgG and free SN-38 showed a mediansurvival of 45 days, comparable to the untreated saline control of 43.5days. A significant and surprising increase in effectiveness of theconjugated, cancer cell targeted antibody-SN-38 conjugate, which wassubstantially more effective than unconjugated antibody and freechemotherapeutic agent alone, was clearly seen. The dose-responsivenessof therapeutic effect of conjugated antibody was also observed. Theseresults demonstrate the clear superiority of the SN-38-antibodyconjugates compared to the combined effect of both unconjugated antibodyand free SN-38 in the same in vivo human lung cancer system.

Example 11 Use of Humanized Anti-TROP-2 IgG-SN-38 Conjugate forEffective Treatment of Diverse Epithelial Cancers

Abstract

The purpose of this study was to evaluate the efficacy of anSN-38-anti-TROP-2 antibody-drug conjugate (ADC) against several humansolid tumor types, and to assess its tolerability in mice and monkeys,the latter with tissue cross-reactivity to hRS7 similar to humans. TwoSN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to theanti-TROP-2-humanized antibody, hRS7. The immunoconjugates werecharacterized in vitro for stability, binding, and cytotoxicity.Efficacy was tested in five different human solid tumor-xenograft modelsthat expressed TROP-2 antigen. Toxicity was assessed in mice and inCynomolgus monkeys.

The hRS7 conjugates of the two SN-38 derivatives were equivalent in drugsubstitution (˜6), cell binding (K_(d)˜1.2 nmol/L), cytotoxicity(IC₅₀˜2.2 nmol/L), and serum stability in vitro (t/_(1/2)˜20 hours).Exposure of cells to the ADC demonstrated signaling pathways leading toPARP cleavage, but differences versus free SN-38 in p53 and p21upregulation were noted. Significant antitumor effects were produced byhRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P≦0.05), Capan-1(P<0.018), BxPC-3 (P<0.005), and COLO 205 tumors (P<0.033) when comparedto nontargeting control ADCs. Mice tolerated a dose of 2×12 mg/kg (SN-38equivalents) with only short-lived elevations in ALT and AST liverenzyme levels. Cynomolgus monkeys infused with 2×0.96 mg/kg exhibitedonly transient decreases in blood counts, although, importantly, thevalues did not fall below normal ranges.

We conclude that the anti-TROP-2 hRS7-CL2A-SN-38 ADC providedsignificant and specific antitumor effects against a range of humansolid tumor types. It was well tolerated in monkeys, with tissue TROP-2expression similar to humans. (Cardillo et al., 2011, Clin Cancer Res17:3157-69.)

Translational Relevance

Successful irinotecan treatment of patients with solid tumors has beenlimited due in large part to the low conversion rate of the CPT-11prodrug into the active SN-38 metabolite. Others have examinednontargeted forms of SN-38 as a means to bypass the need for thisconversion and to deliver SN-38 passively to tumors. We conjugated SN-38covalently to a humanized anti-TROP-2 antibody, hRS7. This antibody-drugconjugate has specific antitumor effects in a range of s.c. human cancerxenograft models, including non-small cell lung carcinoma, pancreatic,colorectal, and squamous cell lung carcinomas, all at nontoxic doses(e.g., ≦3.2 mg/kg cumulative SN-38 equivalent dose).

TROP-2 is widely expressed in many epithelial cancers, but also somenormal tissues, and therefore a dose escalation study in Cynomolgusmonkeys was performed to assess the clinical safety of this conjugate.Monkeys tolerated 24 mg SN-38 equivalents/kg with only minor,reversible, toxicities. Given its tumor-targeting and safety profile,hRS7-SN-38 may provide an improvement in the management of solid tumorsresponsive to irinotecan.

Introduction

Human trophoblast cell-surface antigen (TROP-2), also known as GA733-1(gastric antigen 733-1), EGP-1 (epithelial glycoprotein-1), and TACSTD2(tumor-associated calcium signal transducer), is expressed in a varietyof human carcinomas and has prognostic significance in some, beingassociated with more aggressive disease (see, e.g., Alberti et al.,1992, Hybridoma 11:539-45; Stein et al., 1993, Int J Cancer 55:938-46;Stein et al., 1994, Int J Cancer Suppl. 8:98-102). Studies of thefunctional role of TROP-2 in a mouse pancreatic cancer cell linetransfected with murine TROP-2 revealed increased proliferation in lowserum conditions, migration, and anchorage-independent growth in vitro,and enhanced growth rate with evidence of increased Ki-67 expression invivo and a higher likelihood to metastasize (Cubas et al., 2010, MolCancer 9:253).

TROP-2 antigen's distribution in many epithelial cancers makes it anattractive therapeutic target. Stein and colleagues (1993, Int J Cancer55:938-46) characterized an antibody, designated RS7-3G11 (RS7), thatbound to EGP-1, which was present in a number of solid tumors, but theantigen was also expressed in some normal tissues, usually in a lowerintensity, or in restricted regions. Targeting and therapeuticefficacies were documented in a number of human tumor xenografts usingradiolabeled RS7 (Shih et al., 1995, Cancer Res 55:5857s-63s; Stein etal., 1997, Cancer 80:2636-41; Govindan et al., 2004, Breast Cancer ResTreat 84:173-82), but this internalizing antibody did not showtherapeutic activity in unconjugated form (Shih et al., 1995, Cancer Res55:5857s-63s). However, in vitro it has demonstrated antibody-dependentcellular cytotoxicity (ADCC) activity against TROP-2 positivecarcinomas.

We reported the preparation of antibody-drug conjugates (ADC) using ananti-CEACAM5 (CD66e) IgG coupled to several derivatives of SN-38, atopoisomerase-I inhibitor that is the active component of irinotecan, orCPT-11 (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009,Clin Cancer Res 15:6052-61). The derivatives varied in their in vitroserum stability properties, and in vivo studies found one form(designated CL2) to be more effective in preventing or arresting thegrowth of human colonic and pancreatic cancer xenografts than otherlinkages with more or less stability.

Importantly, these effects occurred at nontoxic doses, with initialtesting failing to determine a dose-limiting toxicity (Govindan et al.,2009, Clin Cancer Res 15:6052-61). These results were encouraging, butalso surprising, because the CEACAM5 antibody does not internalize, aproperty thought to be critical to the success of an ADC. We speculatedthat the therapeutic activity of the anti-CEACAM5-SN-38 conjugate mightbe related to the slow release of SN-38 within the tumor after theantibody localized. Because irinotecan performs best when cells areexposed during the S-phase of their growth cycle, a sustained release isexpected to improve responses. Indeed, SN-38 coupled to nontargeting,plasma extending agents, such as polyethylene glycol (PEG) or micelles,has shown improved efficacy over irinotecan or SN-38 alone (e.g.,Koizumi et al., 2006, Cancer Res 66:10048-56), lending additionalsupport to this mechanism.

Given the RS7 antibody's broad reactivity with epithelial cancers andits internalization ability, we hypothesized that an RS7-SN-38 conjugatecould benefit not only from the sustained release of the drug, but alsofrom direct intracellular delivery. Therefore, we prepared and testedthe efficacy of SN-38 conjugates using a humanized version of the murineRS7 antibody (hRS7). A slight modification was made to the SN-38derivative (Govindan et al., 2009, Clin Cancer Res 15:6052-61), whichimproved the quality of the conjugate without altering its in vitrostability or its efficacy in vivo. This new derivative (designated CL2A)is currently the preferred agent for SN-38 coupling to antibodies.Herein, we show the efficacy of the hRS7-SN-38 conjugate in severalepithelial cancer cell lines implanted in nude mice at nontoxic dosages,with other studies revealing that substantially higher doses could betolerated. More importantly, toxicity studies in monkeys that alsoexpress TROP-2 in similar tissues as humans showed that hRS7-SN-38 wastolerated at appreciably higher amounts than the therapeuticallyeffective dose in mice, providing evidence that this conjugate is apromising agent for treating patients with a wide range of epithelialcancers.

Materials and Methods

Cell Lines, Antibodies, and Chemotherapeutics.

All human cancer cell lines used in this study were purchased from theAmerican Type Culture Collection. These include Calu-3 (non-small celllung carcinoma), SK-MES-1 (squamous cell lung carcinoma), COLO 205(colonic adenocarcinoma), Capan-1 and BxPC-3 (pancreaticadenocarcinomas), and PC-3 (prostatic adenocarcinomas). Humanized RS7IgG and control humanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22(hLL2 IgG, epratuzumab) antibodies were prepared at Immunomedics, Inc.Irinotecan (20 mg/mL) was obtained from Hospira, Inc.

SN-38 Immunoconjugates and In Vitro Aspects.

Synthesis of CL2-SN-38 has been described previously (Moon et al., 2008,J Med Chem 51:6916-26). Its conjugation to hRS7 IgG and serum stabilitywere performed as described (Moon et al., 2008, J Med Chem 51:6916-26;Govindan et al., 2009, Clin Cancer Res 15:6052-61). Preparations ofCL2A-SN-38 (M.W. 1480) and its hRS7 conjugate, and stability, binding,and cytotoxicity studies, were conducted as described previously (Moonet al., 2008, J Med Chem 51:6916-26). Cell lysates were prepared andimmunoblotting for p21^(Waf1/Cip), p53, and PARP (poly-ADP-ribosepolymerase) was performed.

In Vivo Therapeutic Studies.

For all animal studies, the doses of SN-38 immunoconjugates andirinotecan are shown in SN-38 equivalents. Based on a mean SN-38/IgGsubstitution ratio of 6, a dose of 500 μg ADC to a 20-g mouse (25 mg/kg)contains 0.4 mg/kg of SN-38. Irinotecan doses are likewise shown asSN-38 equivalents (i.e., 40 mg irinotecan/kg is equivalent to 24 mg/kgof SN-38). NCr female athymic nude (nu/nu) mice, 4 to 8 weeks old, andmale Swiss-Webster mice, 10 weeks old, were purchased from TaconicFarms. Tolerability studies were performed in Cynomolgus monkeys (Macacafascicularis; 2.5-4 kg male and female) by SNBL USA, Ltd. Animals wereimplanted subcutaneously with different human cancer cell lines. Tumorvolume (TV) was determined by measurements in 2 dimensions usingcalipers, with volumes defined as: L×w²/2, where L is the longestdimension of the tumor and w is the shortest. Tumors ranged in sizebetween 0.10 and 0.47 cm³ when therapy began. Treatment regimens,dosages, and number of animals in each experiment are described in theResults. The lyophilized hRS7-CL2A-SN-38 and control ADC werereconstituted and diluted as required in sterile saline. All reagentswere administered intraperitoneally (0.1 mL), except irinotecan, whichwas administered intravenously. The dosing regimen was influenced by ourprior investigations, where the ADC was given every 4 days or twiceweekly for varying lengths of time (Moon et al., 2008, J Med Chem51:6916-26; Govindan et al., 2009, Clin Cancer Res 15:6052-61). Thisdosing frequency reflected a consideration of the conjugate's serumhalf-life in vitro, to allow a more continuous exposure to the ADC.

Statistics.

Growth curves were determined as percent change in initial TV over time.Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. An f-test was employed to determine equality ofvariance between groups before statistical analysis of growth curves. A2-tailed t-test was used to assess statistical significance between thevarious treatment groups and controls, except for the saline control,where a 1-tailed t-test was used (significance at P≦0.05). Statisticalcomparisons of AUC were performed only up to the time that the firstanimal within a group was euthanized due to progression.

Pharmacokinetics and Biodistribution.

¹¹¹In-radiolabeled hRS7-CL2A-SN-38 and hRS7 IgG were injected into nudemice bearing s.c. SK-MES-1 tumors (˜0.3 cm³). One group was injectedintravenously with 20 μCi (250-μg protein) of ¹¹¹In-hRS7-CL2A-SN-38,whereas another group received 20 μCi (250-μg protein) of ¹¹¹In-hRS7IgG. At various timepoints mice (5 per timepoint) were anesthetized,bled via intracardiac puncture, and then euthanized. Tumors and varioustissues were removed, weighed, and counted by γ scintillation todetermine the percentage injected dose per gram tissue (% ID/g). A thirdgroup was injected with 250 μg of unlabeled hRS7-CL2A-SN-38 3 daysbefore the administration of ¹¹¹In-hRS7-CL2A-SN-38 and likewisenecropsied. A 2-tailed t-test was used to compare hRS7-CL2A-SN-38 andhRS7 IgG uptake after determining equality of variance using the f-test.Pharmacokinetic analysis on blood clearance was performed usingWinNonLin software (Parsight Corp.).

Tolerability in Swiss-Webster Mice and Cynomolgus Monkeys.

Briefly, mice were sorted into 4 groups each to receive 2-mL i.p.injections of either a sodium acetate buffer control or 3 differentdoses of hRS7-CL2A-SN-38 (4, 8, or 12 mg/kg of SN-38) on days 0 and 3followed by blood and serum collection, as described in Results.Cynomolgus monkeys (3 male and 3 female; 2.5-4.0 kg) were administered 2different doses of hRS7-CL2A-SN-38. Dosages, times, and number ofmonkeys bled for evaluation of possible hematologic toxicities and serumchemistries are described in the Results.

Results

Stability and Potency of hRS7-CL2A-SN-38.

Two different linkages were used to conjugate SN-38 to hRS7 IgG. Thefirst is termed CL2-SN-38 and has been described previously (Moon etal., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res15:6052-61). A minor change was made to the synthesis of the CL2 linkerin that the phenylalanine moiety was removed. This change simplified thesynthesis, but did not affect the conjugation outcome (e.g., bothCL2-SN-38 and CL2A-SN-38 incorporated ˜6 SN-38 per IgG molecule).Side-by-side comparisons found no significant differences in serumstability, antigen binding, or in vitro cytotoxicity (not shown).

To confirm that the change in the SN-38 linker from CL2 to CL2A did notimpact in vivo potency, hRS7-CL2A and hRS7-CL2-SN-38 were compared inmice bearing COLO 205 or Capan-1 tumors (not shown), using 0.4 mg or 0.2mg/kg SN-38 twice weekly×4 weeks, respectively, and with starting tumorsof 0.25 cm³ size in both studies. Both the hRS7-CL2A and CL2-SN-38conjugates significantly inhibited tumor growth compared to untreated(AUC_(14 days)P<0.002 vs. saline in COLO 205 model; AUC_(21 days)P<0.001vs. saline in Capan-1 model), and a nontargeting anti-CD20 control ADC,hA20-CL2A-SN-38 (AUC_(14 days)P<0.003 in COLO-205 model; AUC_(35 days):P<0.002 in Capan-1 model). At the end of the study (day 140) in theCapan-1 model, 50% of the mice treated with hRS7-CL2A-SN-38 and 40% ofthe hRS7-CL2-SN-38 mice were tumor-free, whereas only 20% of thehA20-ADC-treated animals had no visible sign of disease. Importantly,there were no differences in efficacy between the 2 specific conjugatesin both the tumor models.

Mechanism of Action.

In vitro cytotoxicity studies demonstrated that hRS7-CL2A-SN-38 had IC₅₀values in the nmol/L range against several different solid tumor lines(Table 8). The IC₅₀ with free SN-38 was lower than the conjugate in allcell lines. Although there was no correlation between TROP-2 expressionand sensitivity to hRS7-CL2A-SN-38, the IC₅₀ ratio of the ADC versusfree SN-38 was lower in the higher TROP-2-expressing cells, most likelyreflecting the enhanced ability to internalize the drug when moreantigen is present.

TABLE 8 Expression of TROP-2 and in vitro cytotoxicity of SN-38 andhRS7-SN-38 in several solid tumor lines TROP-2 Cytotoxicity resultsexpression via FACS hRS7-SN- Median SN-38 95% CI 38 ^(a) 95% CIfluorescence Percent IC₅₀ IC₅₀ IC₅₀ IC₅₀ ADC/free Cell line (background)positive (nmol/L) (nmol/L) (nmol/L) (nmol/L) SN-38 ratio Calu-3 282.2(4.7) 99.6% 7.19 5.77-8.95  9.97  8.12-12.25 1.39 COLO 205 141.5 (4.5)99.5% 1.02 0.66-1.57  1.95 1.26-3.01 1.91 Capan-1 100.0 (5.0) 94.2% 3.502.17-5.65  6.99 5.02-9.72 2.00 PC-3  46.2 (5.5) 73.6% 1.86 1.16-2.99 4.24 2.99-6.01 2.28 SK-MES-1  44.0 (3.5) 91.2% 8.61  6.30-11.76 23.1417.98-29.78 2.69 BxPC-3  26.4 (3.1) 98.3% 1.44 1.04-2.00  4.03 3.25-4.982.80 ^(a) IC₅₀—value is shown as SN-38 equivalents of hRS7-SN-38

SN-38 is known to activate several signaling pathways in cells, leadingto apoptosis. Our initial studies examined the expression of 2 proteinsinvolved in early signaling events (p21^(Waf1/Cip1) and p53) and 1 lateapoptotic event [cleavage of poly-ADP-ribose polymerase (PARP)] in vitro(not shown). In BxPC-3, SN-38 led to a 20-fold increase inp21^(Waf1/Cip1) expression, whereas hRS7-CL2A-SN-38 resulted in only a10-fold increase, a finding consistent with the higher activity withfree SN-38 in this cell line (Table 8). However, hRS7-CL2A-SN-38increased p21^(Waf1/Cip1) expression in Calu-3 more than 2-fold overfree SN-38 (not shown).

A greater disparity between hRS7-CL2A-SN-38- and free SN-38-mediatedsignaling events was observed in p53 expression. In both BxPC-3 andCalu-3, upregulation of p53 with free SN-38 was not evident until 48hours, whereas hRS7-CL2A-SN-38 upregulated p53 within 24 hours (notshown). In addition, p53 expression in cells exposed to the ADC washigher in both cell lines compared to SN-38 (not shown). Interestingly,although hRS7 IgG had no appreciable effect on p21^(Waf1/Cip1)expression, it did induce the upregulation of p53 in both BxPC-3 andCalu-3, but only after a 48-hour exposure. In terms of later apoptoticevents, cleavage of PARP was evident in both cell lines when incubatedwith either SN-38 or the conjugate (not shown). The presence of thecleaved PARP was higher at 24 hours in BxPC-3, which correlates withhigh expression of p21 and its lower IC₅₀. The higher degree of cleavagewith free SN-38 over the ADC was consistent with the cytotoxicityfindings.

Efficacy of hRS7-SN-38.

Because TROP-2 is widely expressed in several human carcinomas, studieswere performed in several different human cancer models, which startedwith an evaluation of the hRS7-CL2-SN-38 linkage, but later, conjugateswith the CL2A-linkage were used. Calu-3-bearing nude mice given 0.04 mgSN-38/kg of the hRS7-CL2-SN-38 every 4 days×4 had a significantlyimproved response compared to animals administered the equivalent amountof hLL2-CL2-SN-38 (TV=0.14±0.22 cm³ vs. 0.80±0.91 cm³, respectively;AUC_(42 days)P<0.026; FIG. 5A). A dose-response was observed when thedose was increased to 0.4 mg/kg SN-38. At this higher dose level, allmice given the specific hRS7 conjugate were “cured” within 28 days, andremained tumor-free until the end of the study on day 147, whereastumors regrew in animals treated with the irrelevant ADC (specific vs.irrelevant AUC_(98 days): P=0.05). In mice receiving the mixture of hRS7IgG and SN-38, tumors progressed >4.5-fold by day 56 (TV=1.10±0.88 cm³;AUC_(56 days)P<0.006 vs. hRS7-CL2-SN-38).

Efficacy also was examined in human colonic (COLO 205) and pancreatic(Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (FIG.5B), hRS7-CL2-SN-38 (0.4 mg/kg, q4dx8) prevented tumor growth over the28-day treatment period with significantly smaller tumors compared tocontrol anti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16±0.09 cm³,1.19±0.59 cm³, and 1.77±0.93 cm³, respectively; AUC_(28 days)P<0.016).The MTD of irinotecan (24 mg SN-38/kg, q2dx5) was as effective ashRS7-CL2-SN-38, because mouse serum can more efficiently convertirinotecan to SN-38 than human serum, but the SN-38 dose in irinotecan(2,400 μg cumulative) was 37.5-fold greater than with the conjugate (64μg total).

Animals bearing Capan-1 showed no significant response to irinotecanalone when given at an SN-38-dose equivalent to the hRS7-CL2-SN-38conjugate (e.g., on day 35, average tumor size was 0.04±0.05 cm³ inanimals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78±0.62 cm³ inirinotecan-treated animals given 0.4 mg/kg SN-38; AUC_(day35)P<0.001;FIG. 5C). When the irinotecan dose was increased 10-fold to 4 mg/kgSN-38, the response improved, but still was not as significant as theconjugate at the 0.4 mg/kg SN-38 dose level (TV=0.17±0.18 cm³ vs.1.69±0.47 cm³, AUC_(day49)P<0.001). An equal dose of nontargetinghA20-CL2-SN-38 also had a significant antitumor effect as compared toirinotecan-treated animals, but the specific hRS7 conjugate wassignificantly better than the irrelevant ADC (TV=0.17±0.18 cm³ vs.0.80±0.68 cm³, AUC_(day49)P<0.018).

Studies with the hRS7-CL2A-SN-38 ADC were then extended to 2 othermodels of human epithelial cancers. In mice bearing BxPC-3 humanpancreatic tumors (FIG. 5D), hRS7-CL2A-SN-38 again significantlyinhibited tumor growth in comparison to control mice treated with salineor an equivalent amount of nontargeting hA20-CL2A-SN-38 (TV=0.24±0.11cm³ vs. 1.17±0.45 cm³ and 1.05±0.73 cm³, respectively;AUC_(day21)P<0.001), or irinotecan given at a 10-fold higher SN-38equivalent dose (TV=0.27±0.18 cm³ vs. 0.90±0.62 cm³, respectively;AUC_(day25)P<0.004). Interestingly, in mice bearing SK-MES-1 humansquamous cell lung tumors treated with 0.4 mg/kg of the ADC (FIG. 5E),tumor growth inhibition was superior to saline or unconjugated hRS7 IgG(TV=0.36±0.25 cm³ vs. 1.02±0.70 cm³ and 1.30±1.08 cm³, respectively;AUC_(28 days), P<0.043), but nontargeting hA20-CL2A-SN-38 or the MTD ofirinotecan provided the same antitumor effects as the specifichRS7-SN-38 conjugate. In all murine studies, the hRS7-SN-38 ADC was welltolerated in terms of body weight loss (not shown).

Biodistribution of hRS7-CL2A-SN-38.

The biodistributions of hRS7-CL2A-SN-38 or unconjugated hRS7 IgG werecompared in mice bearing SK-MES-1 human squamous cell lung carcinomaxenografts (not shown), using the respective ¹¹¹In-labeled substrates. Apharmacokinetic analysis was performed to determine the clearance ofhRS7-CL2A-SN-38 relative to unconjugated hRS7 (not shown). The ADCcleared faster than the equivalent amount of unconjugated hRS7, with theADC exhibiting ˜40% shorter half-life and mean residence time.Nonetheless, this had a minimal impact on tumor uptake (not shown).Although there were significant differences at the 24- and 48-hourtimepoints, by 72 hours (peak uptake) the amounts of both agents in thetumor were similar. Among the normal tissues, hepatic and splenicdifferences were the most striking (not shown). At 24 hourspostinjection, there was >2-fold more hRS7-CL2A-SN-38 in the liver thanhRS7 IgG. Conversely, in the spleen there was 3-fold more parental hRS7IgG present at peak uptake (48-hour timepoint) than hRS7-CL2A-SN-38.Uptake and clearance in the rest of the tissues generally reflecteddifferences in the blood concentration.

Because twice-weekly doses were given for therapy, tumor uptake in agroup of animals that first received a predose of 0.2 mg/kg (250 μgprotein) of the hRS7 ADC 3 days before the injection of the¹¹¹In-labeled antibody was examined. Tumor uptake of¹¹¹In-hRS7-CL2A-SN-38 in predosed mice was substantially reduced atevery timepoint in comparison to animals that did not receive thepredose (e.g., at 72 hours, predosed tumor uptake was 12.5%±3.8% ID/gvs. 25.4%±8.1% ID/g in animals not given the predose; P=0.0123).Predosing had no appreciable impact on blood clearance or tissue uptake(not shown). These studies suggest that in some tumor models, tumoraccretion of the specific antibody can be reduced by the precedingdose(s), which likely explains why the specificity of a therapeuticresponse could be diminished with increasing ADC doses and why furtherdose escalation is not indicated.

Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster Mice and CynomolgusMonkeys.

Swiss-Webster mice tolerated 2 doses over 3 days, each of 4, 8, and 12mg SN-38/kg of the hRS7-CL2A-SN-38, with minimal transient weight loss(not shown). No hematopoietic toxicity occurred and serum chemistriesonly revealed elevated aspartate transaminase (AST) and alaninetransaminase (not shown). Seven days after treatment, AST rose abovenormal levels (>298 U/L) in all 3 treatment groups (not shown), with thelargest proportion of mice being in the 2×8 mg/kg group. However, by 15days posttreatment, most animals were within the normal range. ALTlevels were also above the normal range (>77 U/L) within 7 days oftreatment (not shown) and with evidence of normalization by Day 15.Livers from all these mice did not show histologic evidence of tissuedamage (not shown). In terms of renal function, only glucose andchloride levels were somewhat elevated in the treated groups. At 2×8mg/kg, 5 of 7 mice had slightly elevated glucose levels (range of273-320 mg/dL, upper end of normal 263 mg/dL) that returned to normal by15 days postinjection. Similarly, chloride levels were slightlyelevated, ranging from 116 to 127 mmol/L (upper end of normal range 115mmol/L) in the 2 highest dosage groups (57% in the 2×8 mg/kg group and100% of the mice in the 2×12 mg/kg group), and remained elevated out to15 days postinjection. This also could be indicative of gastrointestinaltoxicity, because most chloride is obtained through absorption by thegut; however, at termination, there was no histologic evidence of tissuedamage in any organ system examined (not shown).

Because mice do not express TROP-2 bound by hRS7, a more suitable modelwas required to determine the potential of the hRS7 conjugate forclinical use. Immunohistology studies revealed binding in multipletissues in both humans and Cynomolgus monkeys (breast, eye,gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas,parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus; not shown). Based on thiscross-reactivity, a tolerability study was performed in monkeys.

The group receiving 2×0.96 mg SN-38/kg of hRS7-CL2A-SN-38 had nosignificant clinical events following the infusion and through thetermination of the study. Weight loss did not exceed 7.3% and returnedto acclimation weights by day 15. Transient decreases were noted in mostof the blood count data (not shown), but values did not fall belownormal ranges. No abnormal values were found in the serum chemistries.Histopathology of the animals necropsied on day 11 (8 days after lastinjection) showed microscopic changes in hematopoietic organs (thymus,mandibular and mesenteric lymph nodes, spleen, and bone marrow),gastrointestinal organs (stomach, duodenum, jejunum, ileum, cecum,colon, and rectum), female reproductive organs (ovary, uterus, andvagina), and at the injection site. These changes ranged from minimal tomoderate and were fully reversed at the end of the recovery period (day32) in all tissues, except in the thymus and gastrointestinal tract,which were trending towards full recovery at this later timepoint.

At the 2×1.92 mg SN-38/kg dose level of the conjugate, there was 1 deatharising from gastrointestinal complications and bone marrow suppression,and other animals within this group showed similar, but more severeadverse events than the 2×0.96 mg/kg group. These data indicate thatdose-limiting toxicities were identical to that of irinotecan; namely,intestinal and hematologic. Thus, the MTD for hRS7-CL2A-SN-38 liesbetween 2×0.96 and 1.92 mg SN-38/kg, which represents a human equivalentdose of 2×0.3 to 0.6 mg/kg SN-38.

Discussion

TROP-2 is a protein expressed on many epithelial tumors, including lung,breast, colorectal, pancreas, prostate, and ovarian cancers, making it apotentially important target for delivering cytotoxic agents. The RS7antibody internalizes when bound to TROP-2 (Shih et al., 1995, CancerRes 55:5857s-63s), which enables direct intracellular delivery ofcytotoxics.

Conjugation of chemotherapeutic drugs to antibodies has been exploredfor over 30 years. Because a substantial portion of an ADC is notprocessed by the tumor, but by normal tissues, there is a risk thatthese agents will be too toxic to normal organ systems before reachingthe therapeutic level in tumors. As with any therapeutic, thetherapeutic window is a key factor determining the potential of an ADC,and thus rather than examining “ultratoxic” drugs, we chose SN-38 as thedrug component of the TROP-2-targeted ADC.

SN-38 is a potent topoisomerase-I inhibitor, with IC₅₀ values in thenanomolar range in several cell lines. It is the active form of theprodrug, irinotecan, that is used for the treatment of colorectalcancer, and which also has activity in lung, breast, and brain cancers.We reasoned that a directly targeted SN-38, in the form of an ADC, wouldbe a significantly improved therapeutic over CPT-11, by overcoming thelatter's low and patient-variable bioconversion to active SN-38.

The Phe-Lys peptide inserted in the original CL2 derivative allowed forpossible cleavage via cathepsin B. In an effort to simplify thesynthetic process, in CL2A, phenylalanine was eliminated, and thus thecathepsin B cleavage site was removed. Interestingly, this product had abetter-defined chromatographic profile compared to the broad profileobtained with CL2 (not shown), but more importantly, this change had noimpact on the conjugate's binding, stability, or potency in side-by-sidetesting. These data suggest that SN-38 in CL2 was released from theconjugate primarily by the cleavage at the pH-sensitive benzyl carbonatebond to SN-38's lactone ring and not the cathepsin B cleavage site.

In vitro cytotoxicity of hRS7 ADC against a range of solid tumor celllines consistently had IC₅₀ values in the nmol/L range. However, cellsexposed to free SN-38 demonstrated a lower IC₅₀ value compared to theADC. This disparity between free and conjugated SN-38 was also reportedfor ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96) and NK012(Koizumi et al., 2006, Cancer Res 66:10048-56). ENZ-2208 utilizes abranched PEG to link about 3.5 to 4 molecules of SN-38 per PEG, whereasNK012 is a micelle nanoparticle containing 20% SN-38 by weight. With ourADC, this disparity (i.e., ratio of potency with free vs. conjugatedSN-38) decreased as the TROP-2 expression levels increased in the tumorcells, suggesting an advantage to targeted delivery of the drug. Interms of in vitro serum stability, both the CL2- and CL2A-SN-38 forms ofhRS7-SN-38 yielded a t/_(1/2) of ˜20 hours, which is in contrast to theshort t/_(1/2) of 12.3 minutes reported for ENZ-2208 (Zhao et al., 2008,Bioconjug Chem 19:849-59), but similar to the 57% release of SN-38 fromNK012 under physiological conditions after 24 hours (Koizumi et al.,2006, Cancer Res 66:10048-56).

Treatment of tumor-bearing mice with hRS7-SN-38 (either with CL2-SN-38or CL2A-SN-38) significantly inhibited tumor growth in 5 different tumormodels. In 4 of them, tumor regressions were observed, and in the caseof Calu-3, all mice receiving the highest dose of hRS7-SN-38 weretumor-free at the conclusion of study. Unlike in humans, irinotecan isvery efficiently converted to SN-38 by a plasma esterase in mice, with agreater than 50% conversion rate, and yielding higher efficacy in micethan in humans. When irinotecan was administered at 10-fold higher orequivalent SN-38 levels, hRS7-SN-38 was significantly better incontrolling tumor growth. Only when irinotecan was administered at itsMTD of 24 mg/kg q2dx5 (37.5-fold more SN-38) did it equal theeffectiveness of hRS7-SN-38. In patients, we would expect this advantageto favor hRS7-CL2A-SN-38 even more, because the bioconversion ofirinotecan would be substantially lower.

We also showed in some antigen-expressing cell lines, such as SK-MES-1,that using an antigen-binding ADC does not guarantee better therapeuticresponses than a nonbinding, irrelevant conjugate. This is not anunusual or unexpected finding. Indeed, the nonbinding SN-38 conjugatesmentioned earlier enhance therapeutic activity when compared toirinotecan, and so an irrelevant IgG-SN-38 conjugate is expected to havesome activity. This is related to the fact that tumors have immature,leaky vessels that allow the passage of macromolecules better thannormal tissues. With our conjugate, 50% of the SN-38 will be released in˜13 hours when the pH is lowered to a level mimicking lysosomal levels(e.g., pH 5.3 at 37° C.; data not shown), whereas at the neutral pH ofserum, the release rate is reduced nearly 2-fold. If an irrelevantconjugate enters an acidic tumor microenvironment, it is expected torelease some SN-38 locally. Other factors, such as tumor physiology andinnate sensitivities to the drug, will also play a role in defining this“baseline” activity. However, a specific conjugate with a longerresidence time should have enhanced potency over this baseline responseas long as there is ample antigen to capture the specific antibody.Biodistribution studies in the SK-MES-1 model also showed that if tumorantigen becomes saturated as a consequence of successive dosing, tumoruptake of the specific conjugate is reduced, which yields therapeuticresults similar to that found with an irrelevant conjugate.

Although it is challenging to make direct comparisons between our ADCand the published reports of other SN-38 delivery agents, some generalobservations can be made. In our therapy studies, the highest individualdose was 0.4 mg/kg of SN-38. In the Calu-3 model, only 4 injections weregiven for a total cumulative dose of 1.6 mg/kg SN-38 or 32 μg SN-38 in a20 g mouse. Multiple studies with ENZ-2208 were done using its MTD of 10mg/kg×5, and preclinical studies with NK012 involved its MTD of 30mg/kg×3. Thus, significant antitumor effects were obtained withhRS7-SN-38 at 30-fold and 55-fold less SN-38 equivalents than thereported doses in ENZ-2208 and NK012, respectively. Even with 10-foldless hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed,whereas lower doses of ENZ-2208 were not presented, and when the NK012dose was lowered 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al.,2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity witha cumulative dose over 1 week of 24 mg/kg SN-38 (1,500 mg/kg of theconjugate), indicating that the MTD was higher. Thus, tumor-bearinganimals were effectively treated with 7.5- to 15-fold lower amounts ofSN-38 equivalents.

As a topoisomerase-I inhibitor, SN-38 induces significant damage to acell's DNA, with upregulation of p53 and p21^(WAF1/Cip1) resulting incaspase activation and cleavage of PARP. When we exposed BxPC-3 andCalu-3 cells to our ADC, both p53 and p21^(WAF1/Cip1) were upregulatedabove basal levels. In addition, PARP cleavage was also evident in bothcell lines, confirming an apoptotic event in these cells. Of interestwas the higher upregulation of p21^(WAF1/Cip1) in BxPC-3 and Calu-3relative to p53 by both free SN-38 and our hRS7-SN-38. This may beindicative of the mutational status of p53 in these 2 cell lines and theuse of a p53-independent pathway for p21^(WAF1/Cip1)-mediated apoptosis.

An interesting observation was the early upregulation of p53 in bothBxPC-3 and Calu-3 at 24 hours mediated by the hRS7-ADC relative to freeSN-38. Even the naked hRS7 IgG could upregulate p53 in these cell lines,although only after a 48-hour exposure. TROP-2 overexpression andcross-linking by antibodies has been linked to several MAPK-relatedsignaling events, as well as intracellular calcium release. Whilebinding of hRS7 was not sufficient to induce apoptosis in BxPC-3 andCalu-3, as evidenced by the lack of PARP cleavage, it may be enough toprime a cell, such that the inclusion of SN-38 conjugated to hRS7 maylead to a greater effect on tumor growth inhibition. Studies arecurrently underway to understand which pathways are involved withhRS7-delivery of SN-38 and how they may differ from free SN-38, and whateffect p53 status may play in this signaling.

Biodistribution studies revealed the hRS7-CL2A-SN-38 had similar tumoruptake as the parental hRS7 IgG, but cleared substantially faster with2-fold higher hepatic uptake, which may be due to the hydrophobicity ofSN-38. With the ADC being cleared through the liver, hepatic andgastrointestinal toxicities were expected to be dose limiting. Althoughmice had evidence of increased hepatic transaminases, gastrointestinaltoxicity was mild at best, with only transient loss in weight and noabnormalities noted upon histopathologic examination. Interestingly, nohematological toxicity was noted. However, monkeys showed an identicaltoxicity profile as expected for irinotecan, with gastrointestinal andhematological toxicity being dose-limiting.

Because TROP-2 recognized by hRS7 is not expressed in mice, it wascritically important to perform toxicity studies in monkeys that have asimilar tissue expression of TROP-2 as humans. Monkeys tolerated 0.96mg/kg/dose (˜12 mg/m²) with mild and reversible toxicity, whichextrapolates to a human dose of ˜0.3 mg/kg/dose (˜11 mg/m²). In a PhaseI clinical trial of NK012, patients with solid tumors tolerated 28 mg/m²of SN-38 every 3 weeks with Grade 4 neutropenia as dose-limitingtoxicity (Hamaguchi et al., 2010, Clin Cancer Res 16:5058-66).Similarly, Phase I clinical trials with ENZ-2208 revealed dose-limitingfebrile neutropenia, with a recommendation to administer 10 mg/m² every3 weeks or 16 mg/m² if patients were administered G-CSF. Because monkeystolerated a cumulative human equivalent dose of 22 mg/m², it is possiblethat even though hRS7 binds to a number of normal tissues, the MTD for asingle treatment of the hRS7 ADC could be similar to that of the othernontargeting SN-38 agents. Indeed, the specificity of the anti-TROP-2antibody did not appear to play a role in defining the DLT, because thetoxicity profile was similar to that of irinotecan. More importantly, ifantitumor activity can be achieved in humans as in mice that respondedwith human equivalent dose of just at 0.03 mg SN-38 equivalents/kg/dose,then significant antitumor responses could be realized clinically.

In conclusion, toxicology studies in monkeys, combined with in vivohuman cancer xenograft models in mice, have indicated that this ADCtargeting TROP-2 is an effective therapeutic in several tumors ofdifferent epithelial origin.

Example 12 Anti-CD22 (Epratuzumab) Conjugated-SN-38 for the Therapy ofHematologic Malgnancies

Abstract

We previously found that slowly internalizing antibodies conjugated withSN-38 could be used successfully when prepared with a linker that allowsapproximately 50% of the IgG-bound SN-38 to dissociate in serum every 24hours. In this study, the efficacy of SN-38 conjugates prepared withepratuzumab (rapidly internalizing) and veltuzumab (slowlyinternalizing), humanized anti-CD22 and anti-CD20 IgG, respectively, wasexamined for the treatment of B-cell malignancies. Both antibody-drugconjugates had similar nanomolar activity against a variety of humanlymphoma/leukemia cell lines, but slow release of SN-38 compromisedpotency discrimination in vitro even against an irrelevant conjugate.When SN-38 was stably linked to the anti-CD22 conjugate, its potency wasreduced 40- to 55-fold. Therefore, further studies were conducted onlywith the less stable, slowly dissociating linker. In vivo, similarantitumor activity was found between CD22 and CD20 antibody-drugconjugate in mice-bearing Ramos xenografts, even though Ramos expressed15-fold more CD20 than CD22, suggesting that the internalization of theepratuzumab-SN-38 conjugate (Emab-SN-38) enhanced its activity.Emab-SN-38 was more efficacious than a nonbinding, irrelevant IgG-SN-38conjugate in vivo, eliminating a majority of well-established Ramosxenografts at nontoxic doses. In vitro and in vivo studies showed thatEmab-SN-38 could be combined with unconjugated veltuzumab for a moreeffective treatment. Thus, Emab-SN-38 is active in lymphoma and leukemiaat doses well below toxic levels and therefore represents a newpromising agent with therapeutic potential alone or combined withanti-CD20 antibody therapy. (Sharkey et al., 2011, Mol Cancer Ther11:224-34.)

Introduction

A significant effort has focused on the biologic therapy of leukemia andlymphoma, where unconjugated antibodies (e.g., rituximab, alemtuzumab,ofatumumab), radioimmunoconjugates (⁹⁰Y-ibritumomab tiuxetan,¹³¹I-tositumomab), and a drug conjugate (gemtuzumab ozogamicin) receivedU.S. Food and Drug Administration (FDA) approval. Another antibody-drugconjugate (ADC), brentuximab vedotin (SGN-35; anti-CD30-auristatin E),recently received accelerated approval by the FDA for Hodgkin lymphomaand anaplastic large-cell lymphomas. There are also a number of otherADCs in preclinical and clinical development that target CD19, CD22,CD37, CD74, and CD79b.

Antibodies against all of these targets are logical choices for carriersof drugs, because they are internalizing. Internalization andspecificity of CD22 have made it a particularly important target forleukemia and lymphomas, with at least 3 different anti-CD22 conjugatesin clinical investigation, including CMC-544 (acid-labile-conjugatedcalicheamicin), an anti-CD22-maytansine conjugate (stably linkedMCC-DM1), and CAT-3888 (formally BL22; a Pseudomonas exotoxinsingle-chain fusion protein). The active agent in all of theseconjugates has subnanomolar potency (i.e., so called ultra-toxics).

We recently developed methods to conjugate antibodies with SN-38, atopoisomerase I inhibitor with low nanomolar potency that is derivedfrom the prodrug, irinotecan (Govindan et al., 2009, Clin Cancer Res15:6052-62; Moon et al., 2008, J Med Chem 51:6916-26). Four SN-38linkage chemistries were examined initially using conjugates preparedwith a slowly internalizing anti-CEACAM5 antibody (Govindan et al.,2009, Clin Cancer Res 15:6052-62; Moon et al., 2008, J Med Chem51:6916-26). The conjugates retained CEACAM5 binding but differed in thedissociation rate of SN-38 in human serum, with half-lives varying fromapproximately 10 to 67 hours (Govindan et al., 2009, Clin Cancer Res15:6052-62). Ultimately, the linker designated CL2, with intermediatestability (˜50% dissociated in 24-35 hours), was selected for furtherdevelopment. CL2 was modified recently, eliminating the phenylalanine inthe cathepsin B-cleavable dipeptide to simplify and improvemanufacturing yields. The new derivative, designated CL2A, retains thepH-sensitive carbonate linkage to the SN-38, but it is no longerselectively cleaved by cathepsin B. Nevertheless, it has identical serumstability and in vivo activity as the original CL2 linker (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). Because significant efficacywithout toxicity was found with the slowly internalizinganti-CEACAM5-SN-38, we postulated that its activity was aided by theslow release of SN-38 from the antibody after it localized in a tumor.Thus, the main objective in this report was to evaluate the therapeuticprospects of conjugates prepared using the CL2A linker with twoantibodies that are highly specific for B-cell cancers but differ intheir antigen expression and internalization properties.

Epratuzumab (Emab) is a rapidly internalizing (e.g., ≧50% within 1hour), humanized anti-CD22 IgG1 that has been evaluated extensively inlymphoma and leukemia in an unconjugated or conjugated form. Veltuzumab(Vmab) is a humanized anti-CD20 antibody that is also being studiedclinically but internalizes slowly (e.g., ˜10% in 1 hour). CD20 isusually expressed at much higher levels than CD22 in non-Hodgkinlymphoma, whereas CD22 is preferentially expressed in acutelymphoblastic leukemia (ALL) but not in multiple myeloma. Bothantibodies are effective in patients as unconjugated agents, but onlyveltuzumab is active in murine xenograft models (Stein et al., 2004,Clin Cancer Res 10:2868-76). On the basis of previous studies thatshowed ⁹⁰Y-Emab combined with unconjugated veltuzumab had enhancedefficacy in NHL models (Mattes et al., 2008, Clin Cancer Res14:6154-60), we also examined the Emab-SN-38+Vmab combination, as thiscould provide additional benefit without competing for the same targetantigen or having additional toxicity.

Materials and Methods

Cell Lines.

Ramos, Raji, Daudi (Burkitt lymphomas), and JeKo-1 (mantle celllymphoma) were purchased from American Type Culture Collection. REH,RS4; 11, MN-60, and 697 (ALL) were purchased from Deutsche Sammlung vonMikroorganismen and Zellkulturen. WSU-FSCCL (follicular NHL) was thegift of Dr. Mitchell R. Smith (Fox Chase Cancer Center, Philadelphia,Pa.). All cell lines were cultured in a humidified CO₂ incubator (5%) at37° C. in recommended supplemented media containing 10 to 20% fetal calfserum and were checked periodically for Mycoplasma.

Antibodies and Conjugation Methods.

Epratuzumab and veltuzumab are humanized anti-CD22 and anti-CD20 IgG1monoclonal antibodies, respectively. Labetuzumab (Lmab), a humanizedanti-CEACAM5 IgG1, and RS7, a humanized anti-TROP-2 antibody (both fromImmunomedics, Inc.), were used as nonbinding, irrelevant controls.Herein, Emab-SN-38, Vmab-SN-38, and Lmab-SN-38 refer to conjugatesprepared using the CL2A linker that was described above. In vitrostudies in human serum showed that approximately 50% of the active SN-38moiety is released from the IgG each day (Cardillo et al., 2011, ClinCancer Res 17:3157-69). Another linker, designated CL2E, is stable inhuman serum over 14 days, but it contains a cathepsin B cleavage site tofacilitate the release of SN-38 when processed in lysosomes. The methodto prepare CL2E and the structures of the CL2A and CL2E linkers aregiven in the Examples above. The conjugates contained approximately 6SN-38 units per IgG (e.g., 1.0 mg of the IgG-SN-38 conjugate contains˜16 μg of SN-38).

In vitro cell binding and cytotoxicity. Flow cytometry was carried outusing the unconjugated specific and irrelevant antibodies incubated for1 hour at 4° C., with binding revealed using fluorescein isothiocyanate(FITC)-Fcγ fragment-specific goat anti-human IgG (JacksonImmunoResearch), also incubated for 1 hour at 4° C. Median fluorescencewas determined on a FACSCALIBUR® flow cytometer (Becton Dickinson) usinga CellQuest software package.

Cytotoxicity was determined using the MTS dye reduction assay (Promega).Dose-response curves [with/without goat anti-human Fcγ F(ab′)₂; JacksonImmunoResearch] were generated from the mean of triplicatedeterminations, and IC₅₀-values were calculated using PRISM® GraphPadsoftware (v5), with statistical comparisons using an F test on the bestfit curves for the data. Significance was set at P<0.05.

Immunoblotting.

After 24- or 48-hour exposure to the test agents, markers of early (p21expression) and late (PARP cleavage) apoptosis were revealed by Westernblotting.

In Vivo Studies.

The subcutaneous Ramos model was initiated by implanting 1×10⁷ cells(0.2 mL) from culture (>95% viability) into 4- to 6-week-old female nudemice (Taconic). Three weeks from implantation, animals with tumorsranging from 0.4 to 0.8 cm³ (measured by caliper, L×W×D) were segregatedinto groups of animals, each with the same range of tumor sizes. Tumorsize and body weights were measured at least once weekly, with animalsremoved from the study when tumors grew to 3.0 cm³ or if theyexperienced 20% or greater body weight loss. The intravenous WSU-FSCCLand 697 models were initiated by intravenous injection of 2.5×10⁶ and1×10⁷ cells, respectively, in female severe combined immunodeficient(SCID) mice (Taconic). Treatment began 5 days after administration ofthe WSU-FSCCL cells and 7 days after the 697 inoculation. Animals wereobserved daily, using hind leg paralysis or other signs of morbidity assurrogate survival endpoints. All treatments were givenintraperitoneally in ≦0.2 mL. The specific dosages and frequency aregiven in the Results section. Because mice convert irinotecan to SN-38efficiently, irinotecan dosing was adjusted on the basis of SN-38equivalents; SN-38 mole equivalents are based on 1.6% of ADC mass and60% of irinotecan mass.

Efficacy was expressed in a Kaplan-Meier curve, using time toprogression (TTP) as surrogate survival endpoints as indicated above.Statistical analysis was conducted by a log-rank test using PRISM®GraphPad software (significance, P<0.05).

Results

Antigen Expression and Cytotoxicity In Vitro.

All cell lines were highly susceptible to SN-38, with EC₅₀ valuesranging from 0.13 nmol/L for Daudi to 2.28 nmol/L for RS4; 11 (Table 9).Except for 697 and RS4; 11, the Emab-SN-38 anti-CD22 conjugate was 2- to7-fold less effective than SN-38. This is a common finding with ourtargeted, as well as other nontargeted, SN-38 conjugates. Despitedifferences in antigen expression, the Emab-SN-38 and Vmab-SN-38 hadsimilar potencies as the nonbinding, Lmab-SN-38 anti-CEACAM5 conjugate,which was likely due to dissociation of approximately 90% of SN-38during the 4-day MTS assay. Other in vitro procedures using shorterexposure times were also ineffective in discriminating differences inthe potencies of conjugates. For example, Annexin V staining after a1-day exposure failed to find differences between untreated and treatedcells (not shown). Upregulation of p21 and PARP cleavage was alsoexamined as early and late markers of apoptosis, respectively. Ramos didnot express p21. However, PARP cleavage was detected, but only after a48-hour exposure, being more strongly expressed in SN-38-treated cells(not shown). The WSU-FSCCL cell line expressed p21, but neither p21upregulation nor PARP cleavage was evident until 48 hours afterEmab-SN-38 exposure. However, both were observed after a 24-hourexposure with free SN-38 (not shown). While the enhanced intensity andearlier activation of apoptotic events with free SN-38 are consistentwith its lower EC₅₀ over the IgG-conjugated form, the results indicatedthat an exposure period of at least 48 hours would be required, but atthis time, approximately 75% of the SN-38 would be released from theconjugate.

TABLE 9 Expression of CD20 and CD22 by FACScan and in vitro cytotoxicityby MTS assay of SN-38 and specific Emab anti-CD22-SN-38, Vmabanti-CD20-SN-38, and Lmab anti- CEACAM5-SN-38 conjugates against severalhematopoietic tumor cell lines CD20 CD22 expression expression EC₅₀values ^(a) Median Median Emab— Vmab— Lmab— Cell fluorescencefluorescence SN-38, 95% SN-38, 95% SN-38, 95% SN-38, 95% line(background) (background) nmol/L CI nmol/L CI nmol/L CI nmol/L CI Raji422.2 (6.8)  45.9 (6.8) 1.42 0.8-2.4 2.10 1.2-3.8 ND — ND — 4.61 2.2-9.5 4.88 2.7-9.0  3.73 1.8-7.6  Ramos 620.4 (4.1)  40.8 (4.1) 0.40 0.2-0.72.92 1.6-5.4 ND — ND — 9.84  4.5-21.6 13.56 4.9-37.2 8.08 2.9-22.2 Daudi815.1 (5.9) 145.0 (5.9) 0.13 0.1-0.2 0.52 0.4-0.7 ND — ND — NHL:follicular WSU-FSCCL  97.4 (4.9)  7.7 (4.9) 0.50 0.3-1.0 0.68 0.4-1.1 ND— ND — NHL: mantle cell 1.05 0.8-1.4  0.83 0.6-1.1  1.17 0.8-1.7  Jeko-1604.6 (6.5)  11.2 (6.5) ND — 2.25 1.3-3.8  1.98 1.1-3.5  2.27 1.3-3.9 ALL: B cell REH  12.3 (4.1)  22.9 (4.1) 0.47 0.3-0.9 1.22 0.8-1.9 ND —ND — 697  6.9 (4.2)  16.0 (4.2) 2.23 1.3-3.9 2.67 1.7-3.7 ND — ND — RS4;11  3.7 (4.1)  23.3 (4.1) 2.28 1.1-4.9 1.68 1.0-3.0 ND — ND — MN-60 21.5 (5.8)  10.3 (5.8) 1.23 0.6-2.1 3.65 2.2-6.2 ND — ND —Abbreviations: CI, confidence interval; ND, not determined. ^(a) EC₅₀expressed as mole equivalents of SN-38 in Emab—SN-38.

We again examined PARP cleavage and p21 expression, this time in cellstreated with Emab-SN-38+Vmab. Confirming the earlier study in Ramos,PARP cleavage first occurs only after a 48-hour exposure to theconjugate, with expression unchanged in the presence of a cross-linkingantibody (not shown). Exposure to veltuzumab for more than 48 hours hadno effect on PARP cleavage, but cleavage was strong within 24 hours whena cross-linking antibody was added (not shown). However, when veltuzumabalone (no cross-linker) was combined with Emab-SN-38, PARP cleavageoccurred after a 24-hour exposure (not shown), indicating veltuzumabcould induce a more rapid onset of apoptosis, even in the absence ofcross-linking. The only notable difference in the WSU-FSCCL cell linewas that the combination greatly enhanced p21 expression at 48 hours(not shown), again suggesting an acceleration of apoptosis inductionwhen veltuzumab is combined with the Emab-SN-38 conjugate. The delay inapoptosis induction in WSU-FSCCL as compared with Ramos is likelyexplained by the lower expression of CD22 and CD20.

Ultratoxic agents often use linkers that are highly stable in serum, astheir premature release would increase toxicity, but these conjugatesmust be internalized for the drug to be delivered optimally. Becauseepratuzumab internalizes rapidly, we examined whether it might benefitfrom a more stably linked SN-38, comparing in vitro cytotoxicity of theCL2A-linked Emab-SN-38 conjugate with the serum-stable CL2E-SN-38conjugate. Both conjugates had a similar binding affinity (not shown),but the more stable Emab-CL2E-SN-38 was approximately 40- to 55-timesless potent than the CL2A conjugate in 3 cell lines (not shown). Whilespecificity was lacking with the CL2A conjugates, the Emab-CL2E-SN-38consistently was approximately two times more potent than the nonbindingLmab-anti-CEACAM5-CL2E-SN-38 conjugate (not shown). We concluded that itwas unlikely that the more stably linked conjugate would be appropriatefor a slowly internalizing veltuzumab conjugate and therefore continuedour investigation only with CL2A-linked SN-38 conjugates.

Because of limitations of the in vitro assays, efficacy was assessed inxenograft models. As indicated in Table 9, all of the lymphoma celllines have much higher expression of CD20 than CD22. Daudi had thehighest expression of CD22 and CD20, but it is very sensitive in vivo tounconjugated veltuzumab and in vitro testing revealed the highestsensitivity to SN-38 (Table 9). These properties would likely make itdifficult to assess differences in activity attributed to the SN-38conjugate versus the unconjugated antibody, particularly whenunconjugated epratuzumab is not an effective therapeutic in animals.Because Ramos had been used previously to show an advantage forcombining ⁹⁰Y-Emab with veltuzumab (Mattes et al., 2008, Clin Cancer Res14:6154-60), we elected to start with a comparison of the Emab-SN-38 andVmab-SN-38 conjugates in the Ramos human Burkitt cell line. Despite flowcytometry showing a 15-fold higher expression of CD20 over CD22,immunohistology of Ramos xenografts showed abundant CD22 and CD20, withCD22 seemingly expressed more uniformly than CD20 (not shown).

Ramos xenografts in untreated animals progressed rapidly, reaching the3.0-cm³ termination size from their starting size of 0.4 cm³ within 6days (not shown), and as reported previously, neither veltuzumab norepratuzumab appreciably affected the progression of well-establishedRamos xenografts (Sharkey et al., 2009, J Nucl Med 50:444-53).Consistent with previous findings using other SN-38 conjugates, none ofthe animals treated with a 4-week, twice-weekly, 0.5 mg/dose treatmentregimen had appreciable weight loss. Both conjugates were highlyeffective in controlling tumor growth, with 80% or more of the animalshaving no evidence of tumor by the end of the 4-week treatment (FIG. 6).The 0.25-mg Vmab-SN-38 dose was better at controlling growth over thefirst 4 weeks, but at 0.5 mg, similar early growth control was observedfor both conjugates. Thus, despite a 15-fold higher expression of CD20than CD22, Emab-SN-38 compared favorably with Vmab-SN-38. Therefore, theremaining studies focused on Emab-SN-38 alone or in combination withunconjugated veltuzumab.

Emab-SN-38 Dose-Response and Specificity.

A dose-response relationship was seen for the specific Emab-SN-38 andirrelevant Lmab-SN-38 conjugates, but Emab-SN-38 had significantlybetter growth control at 2 of the 3 levels tested, and with a strongtrend favoring the specific conjugate at the intermediate dose (FIG. 7).Again, 0.25 mg of Emab-SN-38 ablated a majority of the tumors; here, 7of 10 animals were tumor-free at the end of the 12-week monitoringperiod, with no change in body weight. Animals given irinotecan alone(6.5 μg/dose; approximately the same SN-38 equivalents as 0.25 mg ofconjugate) had a median survival of 1.9 weeks, with 3 of 11 animalstumor-free at the end of the study, which was not significantlydifferent from the 3.45-week median survival for the irrelevantLmab-SN-38 conjugate (P=0.452; FIG. 7C).

In the 697-disseminated leukemia model, the median survival ofsaline-treated animals was just 17 days from tumor inoculation. Animalsgiven unconjugated epratuzumab plus irinotecan (same mole equivalents ofSN-38 as 0.5 mg of the conjugate) had the same median survival, whereasanimals given 0.5 mg of Emab-SN-38 twice weekly starting 7 days fromtumor inoculation survived to 24.5 days, significantly longer thanuntreated animals (P<0.0001) or for unconjugated epratuzumab given withirinotecan (P=0.016). However, Emab-SN-38 was not significantly betterthan the irrelevant conjugate (median survival=22 days; P=0.304), mostlikely reflecting the low expression of CD22 in this cell line.

Emab-SN-38 Combined with Unconjugated Vmab Anti-CD20.

We previously reported improved responses when ⁹⁰Y-Emab was combinedwith unconjugated veltuzumab in the subcutaneous Ramos model (Mattes etal., 2008, Clin Cancer Res 14:6154-60) and thus this possibility wasexamined with Emab-SN-38. In a pilot study, 5 animals bearingsubcutaneous Ramos tumors averaging approximately 0.3 cm³ were givenveltuzumab (0.1 mg), 0.1 mg of Emab-SN-38, or Emab-SN-38+Vmab (allagents given twice weekly for 4 weeks). The median TTP to 2.0 cm³ was22, 14, and more than 77 days, respectively (veltuzumab vs. Emab-SN-38alone, P=0.59; Emab-SN-38+Vmab vs. Emab-SN-38, P=0.0145), providing aninitial indication that the combination of veltuzumab with Emab-SN-38improved the overall therapeutic response. In a follow-up study thatalso used a twice-weekly, 4-week treatment regimen, 6 of 11 animalsgiven 0.1 mg of Emab-SN-38 plus 0.1 mg of veltuzumab had no evidence oftumors 16 weeks from the start of treatment, whereas the median survivalfor animals receiving veltuzumab alone or with 0.1 mg of the controlLmab-SN-38 was 1.9 and 3.3 weeks, respectively, with 3 of 11 animalsbeing tumor-free at 16 weeks in each of these groups (not shown).Despite the longer median TTP and more survivors, no significantdifferences were found between the groups. Thus, in the Ramos model,which has abundant CD20 and moderate levels of CD22, the Emab-SN-38conjugate given at nontoxic dose levels was not significantly betterthan unconjugated anti-CD20 therapy, but the addition of Emab-SN-38 tounconjugated anti-CD20 therapy appeared to improve the response withouttoxicity. It is important to emphasize that the SN-38 conjugates aregiven at levels far less than their maximum tolerated dose, andtherefore these results should not be interpreted that the unconjugatedanti-CD20 therapy is equal to that of the Emab-SN-38 conjugate.

Two additional studies were conducted in an intravenous implanted modelusing the WSU-FSCCL follicular NHL cell line that has a low expressionof CD20 and CD22 (not shown). The median survival time forsaline-treated animals was 40 to 42 days from tumor implantation.Irinotecan alone (not shown), given at a dose containing the same SN-38equivalents as 0.3 mg of the ADC, increased the median survival (49 vs.40 days, respectively; P=0.042), but 14 of 15 animals succumbed todisease progression on day 49, the same day the final 4 of 15 animals inthe saline group were eliminated (not shown). Despite its relatively lowCD20 expression, veltuzumab alone (35 μg twice weekly×4 weeks) waseffective in this model. The median survival increased to 91 days in thefirst study, with 2 cures (day 161), and to 77 days in the second, butwith no survivors after 89 days (veltuzumab alone vs. saline-treated,P<0.001 in both studies). Unconjugated epratuzumab (0.3 mg/dose)combined with irinotecan and veltuzumab had the same median survival asveltuzumab alone, suggesting that neither epratuzumab nor irinotecancontributed to the net response.

As expected because of the low CD22 expression by WSU-FSCCL, Emab-SN-38alone was not as effective as in Ramos. At the 0.15-mg dose, nosignificant benefit over the saline group was seen, but at 0.3 mg, themedian survival increased to 63 days, providing a significantimprovement compared with the saline-treated animals (P=0.006). Thesecond study, using 0.3 mg of Emab-SN-38, confirmed an enhanced survivalcompared with the saline group (75 vs. 40 days; P<0.0001). Thespecificity of this response was not apparent in the first study, wherethe median survival of the irrelevant Lmab-SN-38 conjugate andEmab-SN-38 were not different at either 0.15- or 0.3-mg dose levels (42vs. 49 days and 63 vs. 63 days for the Emab-SN-38 vs. anti-CEACAM5-SN-38conjugates at the 2 doses levels, respectively). However, in the secondstudy, the 0.3-mg dose of Emab-SN-38 provided a significantly improvedsurvival over the irrelevant conjugate (75 vs. 49 days; P<0.0001).Again, the difficulty in showing specificity in this model is mostlikely related to low CD22 expression.

Combining the specific Emab-SN-38 with veltuzumab substantiallyincreases survival, with evidence of more robust responses than thecontrol Lmab-SN-38. For example, in the first study, animals treatedwith veltuzumab plus 0.15 or 0.3 mg of the control conjugate had amedian survival of 98 and 91 days, respectively, which was similar tothat of veltuzumab alone (91 days; not shown). However, veltuzumab plus0.15 mg of the specific Emab-SN-38 conjugate increased the mediansurvival to 140 days. While this improvement was not significantlyhigher than veltuzumab alone (P=0.257), when the Emab-SN-38 dose wasincreased to 0.3 mg with veltuzumab, 6 of 10 animals remained alive atthe end of the study, providing a significant survival advantage overthe control conjugate plus veltuzumab (P=0.0002). In a second study, themedian survival of veltuzumab alone was shorter than in the first (77vs. 91 days), yet the median survival for the control conjugate withveltuzumab was again 91 days, which now yielded a significant survivaladvantage over veltuzumab alone (P<0.0001). Combining the specificEmab-SN-38 conjugate with veltuzumab extended the median survival to 126days, which was significantly longer than the median survival of 75 and77 days for Emab-SN-38 and veltuzumab alone, respectively (P<0.0001 foreach). However, in this study, it did not quite meet the requirementsfor a statistical improvement over the combination with controlanti-CEACAM5-SN-38 conjugate (P=0.078).

Discussion

Over the past 10 years, ADCs have made substantial gains in cancertherapy, yet there also have been some setbacks. The gains occurredlargely when investigators chose to examine agents that were too toxicto be used alone, but when coupled to an antibody, these so-calledultratoxics produced substantially improved responses in preclinicaltesting. The recent approval of brentuximab vedotin, an auristatinconjugate, in Hodgkin lymphoma and the clinical success withtrastuzumab-DM1 anti-HER2-maytansine conjugate as a single agent inbreast cancer refractive to unconjugated trastuzumab suggest that theseADCs bearing ultratoxic agents are becoming accepted treatmentmodalities. However, conjugates prepared with agents that are themselvespotent in the picomolar range can have an increased risk for toxicity,as the recent decision to withdraw gemtuzumab ozogamicin, theanti-CD33-calicheamicin conjugate, from the market suggests (Ravandi,2011, J Clin Oncol 29:349-51). Thus, the success of an ADC may depend onidentifying appropriate chemistries to bind the drug and antibodytogether, as well as defining a suitable target that is sufficientlyexpressed to allow an adequate and selective delivery of the cytotoxicagent.

We developed a linker for coupling SN-38 to IgG that allows SN-38 to bereleased slowly from the conjugate in serum (about 50% per day). Withthis linker, an antibody that is slowly internalized could be aneffective therapeutic, perhaps because the conjugate localized to atumor releases a sufficient amount of drug locally, even without beinginternalized. The CL2A linker also was used recently with an antibody toTROP-2 that was reported to be internalized rapidly (Cardillo et al.,2011, Clin Cancer Res 17:3157-69.). Thus, it appears that the slowrelease mechanism is beneficial for internalizing and noninternalizingantibodies.

In this report, we expanded our assessment of the CL2A linker bycomparing SN-38 conjugates prepared with epratuzumab, a rapidlyinternalizing anti-CD22 IgG, and veltuzumab, a slowly internalizinganti-CD20 IgG, for the treatment of B-cell malignancies. Prior studieswith the murine parent of epratuzumab had indicated that most of theantibody internalizes within 1 hour and 50% of CD22 is reexpressed onthe cell surface within 5 hours (Shih et al., 1994, Int J Cancer56:538-45). This internalization and reexpression process would permitintracellular delivery that might compensate for lower surfaceexpression of CD22. Because many of the B-cell malignancies express muchmore CD20 than CD22, a conjugate targeting CD20 might deliver more molesof drug by releasing its toxic payload after being localized in thetumor.

In vitro cytotoxicity studies could not discriminate the potency of thespecific conjugates or even an irrelevant conjugate because of therelease of SN-38 from the conjugate into the media. Indeed, SN-38 alonewas somewhat more potent than the conjugates, which may reflect itsaccelerated ability to enter the cell and engage topoisomerase I.Because other studies revealed that the conjugates required a 48-hourexposure before early signs of apoptosis could be seen, we concludedthat in vitro testing would not be able to discriminate the potency ofthese 2 conjugates and therefore resorted to in vivo studies.

In xenograft models, both conjugates had similar antitumor activityagainst Ramos tumors, which flow cytometry had indicated expressednearly 15-fold more CD20 than CD22. This lent support to selecting theEmab anti-CD22-SN-38 conjugate especially because it could be combinedwith unconjugated Vmab anti-CD20 therapy without concern that eitheragent would interfere with the binding of the other agent. Indeed, if ananti-CD20-SN-38 conjugate were used, the total IgG protein dose givenlikely would be below a level typically needed for effectiveunconjugated anti-CD20 antibody treatments, as the dose-limitingtoxicity would be driven by the SN-38 content. Adding more unlabeledanti-CD20 to an anti-CD20-SN-38 conjugate would risk reducing theconjugate's uptake and potentially diminishing its efficacy. However, aswe showed previously in combination studies using radiolabeledepratuzumab with unconjugated veltuzumab, benefit can be derived fromboth agents given at their maximum effective and safe dosages. In vitrostudies showed veltuzumab, even in the absence of cross-linking that isused to enhance signaling, accelerated apoptotic events initiated withEmab-SN-38. Thus, as long as the Emab-SN-38 conjugate was as effectiveas the anti-CD20 conjugate, selecting the Emab-SN-38 conjugate is alogical choice because it allows for a more effective combinationtherapy, even in tumors where one or both of the antigens are low inexpression.

Because most ADCs using ultratoxic drugs are stably linked, we alsotested a serum-stable, but intracellularly cleavable, anti-CD22-SN-38conjugate, but determined it was 40- to 55-fold less potent than theCL2A linker. Others have examined a variety of ultratoxic drugsconjugated to anti-CD20 or anti-CD22 antibodies, finding thatinternalizing conjugates are generally more active, but also observingthat even slowly internalizing antibodies could be effective if thereleased drug penetrated the cell membrane. While the CL2A-type linkermay be appropriate for SN-38, it may not be optimal for a more toxicagent, where even a small, sustained release in the serum would increasetoxicity and compromise the therapeutic window.

Emab-SN-38 was active at a cumulative dose of 0.6 mg in mice bearingRamos (75 μg twice weekly for 4 weeks), which extrapolates to a humandose of just 2.5 mg/kg. Thus, Emab-SN-38 should have an ampletherapeutic window in patients. Furthermore, an effective and safe doseof the anti-TROP-2-SN-38 conjugate was combined with a maximum tolerateddose of a ⁹⁰Y-labeled antibody without an appreciable increase intoxicity but with improved efficacy (Sharkey et al., 2011, Mol CancerTher 10:1072-81). Thus, the safety and efficacy profile of these SN-38antibody conjugates are very favorable for other combination therapies.

Even though irinotecan is not used routinely for the treatment ofhematopoietic cancers, SN-38 was as potent in lymphoma and leukemia celllines as in solid tumors (Cardillo et al., 2011, Clin Cancer Res17:3157-69.). In the WSU-FSCCL cell line, the specific and irrelevantIgG conjugates were significantly better than irinotecan, whereas inRamos, the median TTP with the irrelevant conjugate was longer but notsignificantly better than irinotecan. These results are consistent withother studies that have shown that a nonspecific IgG is an excellentcarrier for drugs and more potent in vivo than free drug or conjugatesprepared with albumin or polyethylene glycol (PEG)-Fc. While thePEG-SN-38 conjugate had significant antitumor effects, it was given atits maximum tolerated amounts, ranging from 10 to 30 mg/kg SN-38equivalents (Sapra et al., 2009, Haematologica 94:1456-9). In contrast,the maximum cumulative dose of SN-38 given over 4 weeks to animalsbearing Ramos was only 1.6 mg/kg (i.e., dosing of 0.25 mg of Emab-SN-38given twice weekly over 4 weeks) and this was nontoxic.

The specific therapeutic activity of Emab-SN-38 appeared to improve incell lines with higher CD22 expression. For example, in Ramos, specifictherapeutic effects of Emab-SN-38 alone were recorded at 2 of the 3different dose levels examined, and a sizeable number of tumors werecompletely ablated. In contrast, in WSU-FSCCL that had about 2.5-foldlower expression of CD22, Emab-SN-38 improved survival significantlycompared with the irrelevant anti-CEACAM5-SN-38 conjugate in 1 of 2studies. However, it is important to emphasize that when used incombination with unconjugated anti-CD20 therapy, Emab-SN-38 amplifiesthe therapeutic response. Thus, the combination of these two treatmentscould augment the response even in situations where CD22 is not highlyexpressed.

In conclusion, using the less-stable CL2A-SN-28 linker, Emabanti-CD22-SN-38 conjugate was equally active at nontoxic doses in vivoas a similar anti-CD20-SN-38 conjugate, despite the fact that CD20expression was more than a log-fold higher than CD22. Therapeuticresponses benefited by the combination of Emab-SN-38 with unconjugatedVmab anti-CD20 therapy, even when CD22 expression was low, suggestingthat the combination therapy could improve responses in a number ofB-cell malignancies when both antigens are present The current studiessuggest that this combination is very potent in diverse lymphoma andleukemia preclinical models, yet appears to have less host toxicity.

Example 13 Anti-CD74 (Milatuzumab)SN-38 Conjugates for Treatment ofCD74+ Human Cancers

Abstract

CD74 is an attractive target for antibody-drug conjugates (ADC), becauseit internalizes and recycles after antibody binding. CD74 mostly isassociated with hematological cancers, but is expressed also in solidcancers. Therefore, the utility of ADCs prepared with the humanizedanti-CD74 antibody, milatuzumab, for the therapy CD74-expressing solidtumors was examined. Milatuzumab-doxorubicin and two milatuzumab-SN-38conjugates were prepared with cleavable linkers (CL2A and CL2E),differing in their stability in serum and how they release SN-38 in thelysosome. CD74 expression was determined by flow cytometry andimmunohistology. In vitro cytotoxicity and in vivo therapeutic studieswere performed in the human cancer cell lines A-375 (melanoma), HuH-7and Hep-G2 (hepatoma), Capan-1 (pancreatic), and NCI-N87 (gastric), andRaji Burkitt lymphoma. The milatuzumab-SN-38 ADC was compared to SN-38ADCs prepared with anti-TROP-2 and anti-CEACAM6 antibodies in xenograftsexpressing their target antigens.

Milatuzumab-doxorubicin was most effective in the lymphoma model, whilein A-375 and Capan-1, only the milatuzumab-CL2A-SN-38 showed atherapeutic benefit. Despite much lower surface expression of CD74 thanTROP-2 or CEACAM6, milatuzumab-CL2A-SN-38 had similar efficacy inCapan-1 as anti-TROP-2 CL2A-SN-38, but in NCI-N87, the anti-CEACAM6 andanti-TROP-2 conjugates were superior. Studies in 2 hepatoma cell linesat a single dose level showed significant benefit over saline-treatedanimals, but not against an irrelevant IgG conjugate. CD74 is a suitabletarget for ADCs in some solid tumor xenografts, with efficacy largelyinfluenced by uniformity of CD74 expression, and with CL2A-linked SN-38conjugates providing the best therapeutic responses.

Introduction

CD74, referred to as invariant chain or Ii, is a type II transmembraneglycoprotein that associates with HLA-DR and inhibits the binding ofantigenic peptides to the class II antigen presentation structure. Itserves as a chaperone molecule, directing the invariant chain complexesto endosomes and lysosomes, an accessory molecule in the maturation of Bcells, using a pathway mediated by NF-kB, and in T-cell responses viainteractions with CD44 (Naujokas et al., 1993, Cell 74:257-68), and itis a receptor for the pro-inflammatory cytokine, macrophage migrationinhibitory factor (Leng et al., 2003, J Exp Med 197:1467-76), which isinvolved in activating cell proliferation and survival pathways.

In normal human tissues, CD74 is primarily expressed in B cells,monocytes, macrophages, dendritic cells, Langerhans cells, subsets ofactivated T cells, and thymic epithelium (not shown), and it isexpressed in over 90% of B-cell tumors (Burton et al., 2004, Clin CancerRes 10:6606-11; Stein et al., 2004, Blood 104:3705-11). Early studieshad conflicting data on whether CD74 is present on the membrane, in partbecause the antibodies to the invariant chain were specific for thecytoplasmic portion of the molecule, but also because there arerelatively few copies on the surface, and its half-life on the cellsurface is very short. Approximately 80% of the CD74 on the cell surfaceis associated with the MHC II antigen HLA-DR (Roche et al., 1993, PNASUSA 90:8581-85). Using the murine anti-CD74 antibody, LL1, the RajiBurkitt lymphoma cell line was estimated to have 4.8×10⁴ copies/cell,but because of rapid intracellular transit, ˜8×10⁶ antibody moleculeswere internalized and catabolized per day (Hansen et al., 1996, BiochemJ320:293-300). Thus, CD74 internalization is highly dynamic, with theantibody being moved quickly from the surface and unloaded inside thecell, followed by CD74 re-expression on the surface. Fab′internalization occurs just as rapidly as IgG binding, indicating thatbivalent binding is not required. Later studies with a CDR-graftedversion of murine LL1, milatuzumab (hLL1), found that the antibody couldalter B-cell proliferation, migration, and adhesion molecule expression(Stein et al., 2004, Blood 104:3705-11; Qu et al., 2002, Proc Am AssocCancer Res 43:255; Frolich et al., 2012, Arthritis Res Ther 14:R54), butthe exceptional internalization properties of the anti-CD74 antibodymade it an efficient carrier for the intracellular delivery of cancertherapeutics (e.g., Griffiths et al., 2003, Clin Cancer Res 9:6567-71).Based on preclinical efficacy and toxicology results, Phase I clinicaltrials with milatuzumab-doxorubicin in multiple myeloma (Kaufman et al.,2008, ASH Annual Meeting Abstracts, 112:3697), as well as non-Hodgkinlymphoma and chronic lymphocytic leukemia, have been initiated.

Interestingly, CD74 also is expressed in non-hematopoietic cancers, suchas gastric, renal, urinary bladder, non-small cell lung cancers, certainsarcomas, and glioblastoma (e.g., Gold et al., 2010, Int J Clin ExpPathol 4:1-12), and therefore it may be a therapeutic target for solidtumors expressing this antigen. Since a milatuzumab-doxorubicinconjugate was highly active in models of hematological cancers, it was alogical choice for this assessment. However, we recently developedprocedures for coupling the highly potent topoisomerase I inhibitor,SN-38, to antibodies. SN-38 is the active form of irinotecan, whosepharmacology and metabolism are well known. These conjugates havenanomolar potency in solid tumor cell lines, and were found to be activewith antibodies that were not actively internalized. Prior studiesindicated a preference for a linker (CL2A) that allowed SN-38 todissociate from the conjugate in serum with a half-life of ˜1 day,rather than other linkers that were either more or less stable in serum.However, given milatuzumab's exceptional internalization capability, anew linker that is highly stable in serum, but can release SN-38 whentaken into the lysosome, was developed.

The current investigation examines the prospects for using these threemilatuzumab anti-CD74 conjugates, one with doxorubicin, and two SN-38conjugates, for effective therapy primarily against solid tumors.

Materials and Methods

Human Tumor Cell Lines.

Raji Burkitt lymphoma, A-375 (melanoma), Capan-1 (pancreaticadenocarcinoma), NCI-N87 (gastric carcinoma), Hep-G2 hepatoma and MC/CARmyeloma cell lines were purchased from American Tissue CultureCollection (Manassas, Va.). HuH-7 hepatoma cell line was purchased fromJapan Health Science Research Resources Bank (Osaka, Japan). All celllines were cultured in a humidified CO₂ incubator (5%) at 37° C. inrecommended media containing 10% to 20% fetal-calf serum andsupplements. Cells were passaged <50 times and checked regularly formycoplasma.

Antibodies and Conjugation Methods.

Milatuzumab (anti-CD74 MAb), epratuzumab (anti-CD22), veltuzumab(anti-CD20), labetuzumab (anti-CEACAM5), hMN15 (anti-CEACAM6), and hRS7(anti-TROP-2) are humanized IgG₁ monoclonal antibodies. CL2A and CL2Elinkers and their SN-38 derivatives were prepared and conjugated toantibodies as described in the Examples above. Themilatuzumab-doxorubicin conjugates were prepared as previously described(Griffiths et al., 2003, Clin Cancer Res 9:6567-71). All conjugates wereprepared by disulfide reduction of the IgG, followed by reaction withthe corresponding maleimide derivatives of these linkers.Spectrophotometric analyses estimated the drug:IgG molar substitutionratio was 5-7 (1.0 mg of the protein contains ˜16 μg of SN-38 or 25 μgof doxorubicin equivalent).

In Vitro Cell Binding and Cytotoxicity.

Assays to compare cell binding of the unconjugated and conjugatedmilatuzumab to antigen-positive cells and cytotoxicity testing used theMTS dye reduction method (Promega, Madison, Wis.).

Flow Cytometry and Immunohistology.

Flow cytometry was performed in a manner that provided an assessment ofonly membrane-bound or membrane and cytoplasmic antigen. Immunohistologywas performed on formalin-fixed, paraffin-embedded sections ofsubcutaneous tumor xenografts, staining without antigen retrievalmethods, using antibodies at 10 μg/mL that were revealed with ananti-human IgG conjugate.

In Vivo Studies.

Female nude mice (4-8 weeks old) or female SCID mice (7 weeks old) werepurchased from Taconic (Germantown, N.Y.) and used after a 1-weekquarantine. All agents, including saline controls, were administeredintraperitoneally twice-weekly for 4 weeks. Specific doses are given inResults. Toxicity was assessed by weekly weight measurements. For theRaji Burkitt lymphoma model, SCID mice were injected intravenously with2.5×10⁶ Raji cells in 0.1 mL media. Five days later, animals received asingle intravenous injection (0.1 mL) of the conjugate or saline(N=10/group). Mice were observed daily for signs of distress andparalysis, and were euthanized when either hind-limb paralysisdeveloped, >15% loss of initial weight, or if otherwise moribund(surrogate survival endpoints).

Subcutaneous tumors were measure by caliper in two dimensions, and thetumor volume (TV) calculated as L×w²/2, where L is the longest diameterand w is the shortest. Measurements were made at least once weekly, withanimals terminated when tumors grew to 1.0 cm³ (i.e., surrogate survivalend-point). The A-375 melanoma cell line (6×10⁶ cells in 0.2 mL) wasimplanted in nude mice and therapy was initiated when tumors averaged0.23±0.06 cm³ (N=8/group). Capan-1 was implanted subcutaneously in nudemice using a combination of tumor suspension from serially-passagedtumors (0.3 mL of a 15% w/v tumor suspension) combined with 8×10⁶ cellsfrom tissue culture. Treatments were initiated when TV averaged0.27±0.05 cm³ (N=10/group). NCI-N87 gastric tumor xenografts wereinitiated by injecting 0.2 mL of a 1:1 (v/v) mixture of matrigel and1×10⁷ cells from terminal culture subcutaneously. Therapy was startedwhen the TV averaged 0.249±0.045 cm³ (N=7/group). The same procedure wasfollowed for developing the Hep-G2 and HuH-7 hepatoma xenografts in nudemice. Therapy was started when Hep-G2 averaged 0.364±0.062 cm³(N=5/group) and HuH-7 averaged 0.298±0.055 cm³ (N=5/group).

Efficacy is expressed in Kaplan-Meier survival curves, using thesurrogate end-points mentioned above for determining the median survivaltimes. Analysis was performed by a log-rank (Mantel-Cox) test usingPrism GraphPad software (LaJolla, Calif.), with significance at P<0.05.

Results

CD74 Expression in Human Tumor Cell Lines and Xenografts.

Six cell lines derived from 4 different solid tumor types wereidentified as CD74-positive based primarily on the analysis ofpermeabilized cells (Table 10), since the MFI of membrane-only CD74 inthe solid tumor cell lines very often was <2-fold higher than thebackground MFI (except A-375 melanoma cell line). Surface CD74expression in Raji was >5-fold higher than the solid tumor cell lines,but total CD74 in permeabilized Raji cells was similar to most of thesolid tumor cell lines.

TABLE 10 CD74 expression by flow cytometry expressed as mean fluorescentintensity (MFI) of milatuzumab-positive gated cells. Surface and Surfacecytoplasmic hLL1 MFI Ratio hLL1 MFI Ratio Cell line (bkgd)^(a) hLL1:bkgd(bkgd)^(b) hLL1:bkgd Panc CA^(c) Capan-1 22 (12) 1.8 248 (5) 49.6Gastric Hs746T 17 (8)  2.1 144 (5) 28.8 NCI-N87 5 (4) 1.3 220 (6) 36.7Melanoma A-375 16 (3)  5.3 185 (6) 30.8 Hepatoma Hep-G2 9 (6) 1.5 156(5) 31.2 HuH-7 8 (5) 1.6 114 (4) 28.5 Lymphoma Raji 59 (3)  19.6 143 (5)28.6 ND, not done ^(a)Background MFI of cells incubated with GAH-FITConly.

Immunohistology showed Raji subcutaneous xenografts had a largelyuniform and intense staining, with prominent cell surface labeling (notshown). The Hep-G2 hepatoma cell line had the most uniform uptake of thesolid tumors, with moderately strong, but predominantly cytoplasmic,staining (not shown), followed by the A-375 melanoma cell line that hadsomewhat less uniform staining with more intense, yet mostlycytoplasmic, expression (not shown). The Capan-1 pancreatic (not shown)and NCI-N87 (not shown) gastric carcinoma cell lines had moderate(Capan-1) to intense (NCI-N87) CD74 staining, but it was not uniformlydistributed. The HuH-7 hepatoma cell line (not shown) had the leastuniform and the weakest staining.

Immunoreactivity of the Conjugates.

K_(d) values for unconjugated milatuzumab, milatuzumab-CL2A- andCL2E-SN-38 conjugates were not significantly different, averaging 0.77nM, 0.59 nM, and 0.80 nM, respectively. K_(d) values for theunconjugated and doxorubicin-conjugated milatuzumab measured in theMC/CAR multiple myeloma cell line were 0.5±0.02 nM and 0.8±0.2 nM,respectively (Sapra et al., 2008, Clin Cancer Res 14:1888-96).

In Vitro Drug Release and Serum Stabilities of Conjugates.

The release mechanisms of SN-38 from the mercaptoethanol-capped CL2A andCL2E linkers were determined in an environment partially simulatinglysosomal conditions, namely, low pH (pH 5.0), and in the presence orabsence of cathepsin B. The CL2E-SN-38 substrate was inert at pH 5 inthe absence of the enzyme (not shown), but in the presence of cathepsinB, cleavage at the Phe-Lys site proceeded quickly, with a half-life of34 min (not shown). The formation of active SN-38 requiresintramolecular cyclization of the carbamate bond at the 10^(th) positionof SN-38, which occurred more slowly, with a half-life of 10.7 h (notshown).

As expected, cathepsin B had no effect on the release of active SN-38 inthe CL2A linker. However, CL2A has a cleavable benzyl carbonate bond,releasing active SN-38 at a rate similar to the CL2E linker at pH 5.0,with a half-life of ˜10.2 h (not shown). The milatuzumab-doxorubicinconjugate, which has a pH-sensitive acylhydrazone bond, had a half-lifeof 7 to 8 h at pH 5.0 (not shown).

While all of these linkers release the drug at relatively similar ratesunder lysosomally-relevant conditions, they have very differentstabilities in serum. Milatuzumab-CL2A-SN-38 released 50% of free SN-38in 21.55±0.17 h (not shown), consistent with other CL2A-SN-38conjugates. The CL2E-SN-38 conjugate, however, was highly inert, with ahalf-life extrapolated to ˜2100 h. The milatuzumab-doxorubicin conjugatereleased 50% of the doxorubicin in 98 h, which was similar to 2 otherantibody-doxorubicin conjugates (not shown).

Cytotoxicity.

A significant issue related to the evaluation of these conjugates wasthe relative potency of free doxorubicin and SN-38 in hematopoietic andsolid tumor cell lines. Our group previously reported that SN-38 wasactive in several B-cell lymphoma and acute leukemia cell lines, withpotencies ranging from 0.13 to 2.28 nM (Sharkey et al., 2011, Mol CancerTher 11:224-34). SN-38 potency in 4 of the solid tumor cell lines thatwere later used for in vivo therapy studies ranged from 2.0 to 6 nM (notshown). Doxorubicin had a mixed response, with 3-4 nM potency in theRaji lymphoma and the A-375 melanoma cell lines, but it was nearly 10times less potent against Capan-1, NCI-N87, and Hep G2 cell lines. Otherstudies comparing the potency of SN-38 to doxorubicin found: LS174Tcolon cancer, 18 vs. 18 (nM potency of SN-38 vs. doxorubicin,respectively); MDA-MB-231 breast cancer, 2 vs. 2 nM; SK-OV-4 ovariancancer, 18 vs. 90 nM; Calu-3 lung adenocarcinoma, 32 vs. 582 nM; Capan-2pancreatic cancer, 37 vs. 221 nM; and NCI-H466 small cell lung cancer,0.1 vs. 2 nM. Thus, SN-38 was 5- to 20-fold more potent than doxorubicinin 4 of these 6 cell lines, with similar potency in LS174T andMDA-MB-231. Collectively, these data indicate that doxorubicin is lesseffective against solid tumors than SN-38, while SN-38 appears to beequally effective in solid and hematopoietic tumors.

As expected, the 3 conjugate forms were often some order of magnitudeless potent than the free drug in vitro, since both drugs are expectedto be transported readily into the cells, while drug conjugates requireantibody binding to transport drug inside the cell (not shown). TheCL2A-linked SN-38 conjugate is an exception, since more than 90% of theSN-38 is released from the conjugate into the media over the 4-day assayperiod (Cardillo et al., 2011, Clin Cancer Res 17:3157-69; Sharkey etal., 2011, Mol Cancer Ther 11:224-34). Thus, even if the conjugate wasinternalized rapidly, it would be difficult to discern differencesbetween the free drug and the CL2A-linked drug.

The stable CL2E-linked SN-38 performed comparatively well in the Rajicell line, compared to free SN-38, but it had substantially (7- to16-fold) lower potency in the 4 solid tumor cell lines, suggesting therelatively low surface expression of CD74 may be playing a role inminimizing drug transport in these solid tumors. Themilatuzumab-doxorubicin conjugate had substantial differences in itspotency when compared to the free doxorubicin in all cell lines, whichwas of similar magnitude as the CL2E-SN-38 conjugates to free SN-38 inthe solid tumor cell lines.

In the 6 additional cell lines mentioned above, themilatuzumab-CL2A-SN-38 conjugate was 9- to 60-times more potent than themilatuzumab-doxorubicin conjugate (not shown), but again, this resultwas influenced largely by the fact that the CL2A-linked conjugatereleases most of its SN-38 into the media over the 4-day incubationperiod, whereas the doxorubicin conjugate would at most release 50% ofits drug over this same time. The CL2E-linked milatuzumab was notexamined in these other cell lines.

In Vivo Therapy of Human Tumor Xenografts.

Previous in vivo studies with the milatuzumab-doxorubicin or SN-38conjugates prepared with various antibodies had indicated they wereefficacious at doses far lower than their maximum tolerated dose(Griffiths et al., 2003, Clin Cancer Res 9:6567-71; Sapra et al., 2005,Clin Cancer Res 11:5257-64; Govindan et al., 2009, Clin Cancer Res15:6052-61; Cardillo et al., 2011, Clin Cancer Res 17:3157-69; Sharkeyet al., 2011, Mol Cancer Ther 11:224-34), and thus in vivo testingfocused on comparing similar, but fixed, amounts of each conjugate atlevels that were well-tolerated.

Initial studies first examined the doxorubicin and SN-38 conjugates in adisseminated Raji model of lymphoma in order to gauge how themilatuzumab-doxorubicin conjugate compared to the 2 SN-38 conjugates(not shown). All specific conjugates were significantly better thannon-targeting labetuzumab-SN-38 conjugate or saline-treated animals,which had a median survival of only 20 days (P<0.0001). Despite in vitrostudies indicating as much as an 8-fold advantage for the SN-38conjugates in Raji, the best survival was seen with themilatuzumab-doxorubicin conjugates, where all animals given a single17.5 mg/kg (350 μg) dose and 7/10 animals given 2.0 mg/kg (40 μg) werealive at the conclusion of the study (day 112) (e.g., 17.5 mg/kg dosemilatuzumab-doxorubicin vs. milatuzumab-CL2A-SN-38, P=0.0012). Survivalwas significantly lower for the more stable CL2E-SN-38 conjugates(P<0.0001 and P=0.0197, 17.5 and 2.0 mg/kg doses for the CL2A vs. CL2E,respectively), even though in vitro studies suggested that bothconjugates would release active SN-38 at similar rates wheninternalized.

Five solid tumor cell lines were examined, starting with the A-375melanoma cell line, since it had the best in vitro response to bothdoxorubicin and SN-38. A-375 xenografts grew rapidly, withsaline-treated control animals having a median survival of only 10.5days (not shown). A 12.5 mg/kg (0.25 mg per animal) twice-weekly dose ofthe milatuzumab-CL2A-SN-38 conjugate extended survival to 28 days(P=0.0006), which was significantly better than the controlepratuzumab-CL2A-SN-38 conjugate having a median survival of 17.5 days(P=0.0089), with the latter not being significantly different from thesaline-treated animals (P=0.1967). The milatuzumab-CL2A conjugateprovided significantly longer survival than the milatuzumab-CL2E-SN-38conjugate (P=0.0014), which had the same median survival of 14 days asits control epratuzumab-CL2E-SN-38 conjugate. Despite giving a 2-foldhigher dose of the milatuzumab-doxorubicin than the SN-38 conjugates,the median survival was no better than the saline-treated animals (10.5days).

As with the A-375 melanoma model, in Capan-1, only the CL2A-linked SN-38conjugate was effective, with a median survival of 35 days,significantly different from untreated animals (P<0.036) (not shown),even at a lower dose (5 mg/kg; 100 μg per animal) (P<0.02). Neither themilatuzumab-CL2E nor the non-targeting epratuzumab-CL2A-SN-38conjugates, or a 2-fold higher dose of the milatuzumab-doxorubicinconjugate, provided any survival advantage (P=0.44 vs. saline). It isnoteworthy that in the same study with animals given the same dose ofthe internalizing anti-TROP-2 CL2A-SN-38 conjugate (hRS7-SN-38;IMMU-132), the median survival was equal to milatuzumab-CL2A-SN-38 (notshown). The hRS7-CL2A-SN-38 conjugate had been identified previously asan ADC of interest for treating a variety of solid tumors (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). The MFI for surface-binding hRS7on Capan-1 was 237 (not shown), compared to 22 for milatuzumab (seeTable 10). Thus, despite having a substantially lower surface antigenexpression, the milatuzumab-CL2A-SN-38 conjugate performed as well asthe hRS7-CL2A-SN-38 conjugate in this model.

With the milatuzumab-doxorubicin conjugate having inferior therapeuticresults in 2 of the solid tumor xenografts, the focus shifted to comparethe milatuzumab-SN-38 conjugates to SN-38 conjugates prepared with otherhumanized antibodies against TROP-2 (hRS7) or CEACAM6 (hMN-15), whichare more highly expressed on the surface of many solid tumors(Blumenthal et al., 2007, BMC Cancer 7:2; Stein et al., 1993, Int JCancer 55:938-46). Three additional xenograft models were examined.

In the gastric tumor model, NCI-N87, animals given 17.5 mg/kg/dose (350μg) of milatuzumab-CL2A-SN-38 provided some improvement in survival, butit failed to meet statistical significance compared to thesaline-treated animals (31 vs. 14 days; P=0.0760) or to the non-bindingveltuzumab anti-CD20-CL2A-SN39 conjugate (21 days; P=0.3128) (notshown). However, the hRS7- and hMN-15-CL2A conjugates significantlyimproved the median survival to 66 and 63 days, respectively (P=0.0001).The MFI for surface-expressed TROP-2 and CEACAM6 were 795 and 1123,respectively, much higher than CD74 that was just 5 (see Table 10).Immunohistology showed a relatively intense cytoplasmic expression ofCD74 in the xenograft of this cell line, but importantly it wasscattered, appearing only in defined pockets within the tumor (notshown). CEACAM6 and TROP-2 were more uniformly expressed than CD74 (notshown), with CEACAM6 being more intensely present both cytoplasmicallyand on the membrane, and TROP-2 primarily found on the membrane. Thus,the improved survival with the anti-CEACAM6 and anti-TROP-2 conjugatesmost likely reflects both higher antigen density and more uniformexpression in NCI-N87.

In the Hep-G2 hepatoma cell line (not shown), immunohistology showed avery uniform expression with moderate cytoplasmic staining of CD74, andflow cytometry indicated a relatively low surface expression (MFI=9).The MFI with hMN-15 was 175 and immunohistology showed a fairly uniformmembrane and cytoplasmic expression of CEACAM6, with isolated pockets ofvery intense membrane staining (not shown). A study in animals bearingHep-G2 xenografts found the milatuzumab-CL2A-SN-38 extended survival to45 days compared to 21 days in the saline-treated group (P=0.0048),while the hMN-15-CL2A-SN-38 conjugate improved survival to 35 days.There was a trend favoring the milatuzumab conjugate overhMN-15-CL2A-SN-38, but it did not achieve statistical significance (46vs. 35 days; P=0.0802). However, the non-binding veltuzumab-CL2A-SN-38conjugate provided a similar survival advantage as the milatuzumabconjugate. We previously observed therapeutic results with non-bindingconjugates could be similar to the specific CL2A-linked conjugate,particularly at higher protein doses, but titration of the specific andcontrol conjugates usually revealed selectively. Thus, neither of thespecific conjugates provided a selective therapeutic advantage at thesedoses in this cell line.

Another study using the HuH-7 hepatoma cell line (not shown), which hadsimilar surface expression, but slightly lower cytoplasmic levels asHep-G2 (see Table 10), found the hMN-15-SN-38 conjugate providing alonger (35 vs. 18 days), albeit not significantly different, survivaladvantage than the milatuzumab-CL2A conjugate (P=0.2944). While both thehMN-15 and milatuzumab conjugates were significantly better than thesaline-treated animals (P=0.008 and 0.009, respectively), again, neitherconjugate was significantly different from the non-targetedveltuzumab-SN-38 conjugate at this dose level (P=0.4602 and 0.9033,respectively). CEACAM6 surface expression was relatively low in thiscell line (MFI=81), and immunohistology showed that both CD74 (notshown) and CEACAM6 (not shown) were very faint and highly scattered.

Discussion

The antibody-drug conjugate (ADC) approach for tumor-selectivechemotherapy is an area of considerable current interest (e.g., Govindanet al., 2012, Expert Opin Biol Ther 12:873-90; Sapra et al., 2011,Expert Opin Biol Ther 20:1131-49. The recent clinical successes (Pro etal., 2012, Expert Opin Biol Ther 12:1415-21; LoRusso et al., 2011, ClinCancer Res 17:437-47) have occurred in a large part with the adoption ofsupertoxic drugs in place of the conventional chemotherapeutic agentsthat had been used previously. However, target selection, the antibody,and the drug linker are all factors that influence optimal performanceof an ADC. For example, in the case of trastuzumab-DM1, HER2 is abundanton tumors expressing this antigen, the antibody is internalized, and theantibody itself has anti-tumor activity, all of which could combine toenhance therapeutic outcome. In stark contrast, CD74 is expressed at amuch lower level on the surface of cells, but its unique internalizationand surface re-expression properties has allowed a milatuzumab anti-CD74ADC to be effective in hematopoietic cancer xenograft models even with amoderately toxic drug, such as doxorubicin (Griffiths et al., 2003, ClinCancer Res 9:6567-71; Sapra et al., 2005, Clin Cancer Res 11:5257-64).Although doxorubicin is used more frequently in hematopoietic cancers,while SN-38 and other camptothecins are administered to patients withsolid tumors, we decided to assess the utility of doxorubicin and SN-38conjugates of milatuzumab in solid tumors. The milatuzumab-doxorubicinconjugate was effective in xenograft models of various hematologicalcancers, leading to its clinical testing (NCT01101594 and NCT01585688),while several SN-38 conjugates were effective in solid and hematologicaltumor models, leading to 2 new SN-38 conjugates being pursued in Phase Iclinical trials of colorectal and diverse epithelial cancers(NCT01270698 and NCT01631552).

In vitro, unconjugated doxorubicin and SN-38 had similar potency asdoxorubicin against the Raji lymphoma cell line, but SN-38 was morepotent in a number of different solid tumor cell lines. Interestingly,in vivo, the milatuzumab-doxorubicin conjugate provided the bestresponse in Raji as compared to the milatuzumab-SN-38 conjugates.However, in Capan-1 and A-375, milatuzumab-doxorubicin was lesseffective than the CL2A-linked SN-38 milatuzumab conjugate, even thoughin vitro testing had indicated that A-375 was equally sensitive to freedoxorubicin as to free SN-38. Two other cell lines, MDA-MB-231 breastcancer and LS174T colon cancer, also had similar potency with freedoxorubicin as SN-38 in vitro, but since in vitro testing indicatedSN-38 was equally effective in solid and hematological cancers, and withSN-38 having a 5- to 20-fold higher potency than doxorubicin in mostsolid tumor cell lines evaluated, we decided to focus on the 2milatuzumab-SN-38 conjugates for solid tumor therapy. However, to bettergauge the utility of the milatuzumab-SN-38 conjugates, we included acomparative assessment to SN-38 ADCs prepared with antibodies againstother antigens that are present in a variety of solid tumors.

We previously had investigated therapeutic responses with theinternalizing hRS7 anti-TROP-2 CL2A-linked SN-38 conjugate in theCapan-1 cell line (Cardillo et al., 2011, Clin Cancer Res 17:3157-69),and therefore the efficacy of milatuzumab and hRS7 SN-38 conjugates werecompared. In this study, both conjugates significantly improved survivalcompared to control antibodies, with the CL2A-linked SN-38 conjugates ofeach being superior to the CL2E-linked conjugates. Since flow cytometryhad indicated TROP-2 expression was higher than CD74 in Capan-1, thisresult suggested that the transport capabilities of CD74, which wereknown to be exceptional, were more efficient than TROP-2. However, it iswell known that antigen accessibility (i.e., membrane vs. cytoplasm,physiological and “binding-site” barriers) and distribution among cellswithin a tumor are critical factors influencing every form of targetedtherapy, particularly those that depend on adequate intracellulardelivery of a product to individual cells (Thurber et al., 2008, AdvDrug Del Rev 60:1421-34). In situations where the antigen is notuniformly expressed in all cells within the tumor, having a targetedagent that slowly releases its payload after localizing in the tumor,such as the CL2A-linked conjugates, would allow the drug to diffuse tonon-targeted bystander cells, thereby enhancing its efficacy range.Indeed, high antigen expression could potentially impede tumorpenetration as per the binding-site barrier effect, but theextracellular release mechanism could provide a mechanism for the drugto diffuse within the tumor. This mechanism also is thought to aid theefficacy of other conjugates that we have examined using poorlyinternalizing antibodies, such as anti-CEACAM5 and the anti-CEACAM6 usedherein. Conjugates based on milatuzumab rely more heavily on theantibody's direct interaction with the tumor cell, taking advantage ofCD74's rapid internalization and re-expression that can compensate forits lower abundance on the surface of cells. However, this advantagewould be mitigated when CD74 is highly scattered within the tumor, andwithout a mechanism to retain the conjugate within the tumor, thebenefit of the drug's slow release from the conjugate would be lost. Aprevious review of human gastrointestinal tumors by our group suggeststhat they often have a high level of expression with good uniformity(Gold et al., 2010, Int J Clin Exp Pathol 4:1-12).

During our initial assessment of suitable linkers for SN-38, a number ofdifferent derivatives were examined, including a ‘CL2E’-like linker thatwas designed to be coupled at the 20-hydroxyl position of SN-38, similarto the CL2A linker. However, that antibody conjugate lacked sufficientantitumor activity and was not pursued. Given the exceptionalinternalization properties of milatuzumab, we decided to revisit theSN-38-linker chemistry, with the hypothesis that the rapidinternalization of a CD74 conjugate would enhance drug loading of a morestable conjugate. We surmised that if the leaving group was phenolic,this could promote cyclization, and therefore, the CL2E-linker wasdesigned to join at the phenolic 10-position of SN-38.

At the onset, the CL2E-linked SN-38 conjugate had a promisingly similarIC₅₀ as the CL2A conjugate in the Raji cell line, which was consistentwith the view that if rapidly internalized, both conjugates wouldrelease the active form of SN-38 at approximately the same rate.However, as already mentioned, the in vitro activity of the CL2Aconjugate is influenced largely by the release of SN-38 into the media,and does not necessarily reflect uptake by the intact conjugate. Whenthe CL2E-linked conjugate was found to be much less potent in the solidtumor cell lines than the CL2A conjugate, this suggested that the lowersurface expression of CD74 affected the internalization of SN-38 viamilatuzumab binding. However, when in vivo studies in Raji showed themilatuzumab-CL2A-SN-38 was superior to the CL2E conjugate, some otherfactor had to be considered that would affect CL2E's efficacy. Onepossible explanation is that the linker design in CL2E-SN-38 leaves the20-position of the drug underivatized, rendering the lactone groupsusceptible to ring-opening. Indeed, studies with irinotecan have shownSN-38's potency is diminished by a number of factors, with the lactonering opening to the carboxylate form possessing only 10% of the potencyof the intact lactone form. In contrast, the CL2A-linked SN-38 isderivatized at the 20-hydroxyl position, a process that stabilizes thelactone group in camptothecins under physiological conditions.Therefore, SN-38's lactone ring is likely protected from cleavage in theCL2A, but not the CL2E conjugate. Thus, the destabilization of thelactone ring could have contributed to CL2E's diminished efficacy invivo. Since the in vitro stability studies and the analysis of serumstability were performed under acidic conditions, we do not have adirect measure of the carboxylate form of SN-38 in either of theseconjugates.

In conclusion, in vitro and in vivo results indicate that themilatuzumab-doxorubicin conjugate is superior to the CL2A-SN-38conjugate in the Raji lymphoma cell line, which may reflect the improvedstability of the doxorubicin conjugate compared to the CL2A one.However, the finding that the CL2A-SN-38 conjugate was more effectivethan the highly stable CL2E-SN-38 conjugate suggests that other issues,potentially related to activation of the drug or cell linesensitivities, may be at play.

CD74 has multiple roles in cell biology; in antigen-presenting cells, itmay have a more dominant role in processing antigenic peptides, where issolid tumors, its role might be related more to survival. Thesedifferent roles could affect intracellular trafficking and processing.Alternatively, the lower efficacy of the CL2E-linked SN-38 could reflectdrug inactivation by lactone ring-opening in SN-38, implicating theimportance of the specific linker. Finally, in the solid tumor models,antigen accessibility appears to have a dominant role in definingmilatuzumab-CL2A-SN-38's potency when measured against conjugatesprepared with other internalizing (hRS7) or poorly internalizingantibodies (hMN15) that were more accessible (surface expressed) andabundant. We suspect this finding is universal for targeted therapies,but these studies have at least shown that the unique internalizationproperties of a CD74-targeted agent can provide significant efficacyeven when surface expression of the target antigen is minimal.

Example 14 Use of hRS7-SN-38 (IMMU-132) to Treat Therapy-RefractiveMetastatic Colonic Cancer (mCRC)

The patient was a 62-year-old woman with mCRC who originally presentedwith metastatic disease in January 2012. She had laparoscopic ilealtransverse colectomy as the first therapy a couple of weeks afterdiagnosis, and then received 4 cycles of FOLFOX (leucovorin,5-fluorouracil, oxaliplatin) chemotherapy in a neoadjuvant setting priorto right hepatectomy in March 2012 for removal of metastatic lesions inthe right lobe of the liver. This was followed by an adjuvant FOLFOXregimen that resumed in June, 2012, for a total of 12 cycles of FOLFOX.In August, oxaliplatin was dropped from the regimen due to worseningneurotoxicity. Her last cycle of 5-FU was on Sep. 25, 2012.

CT done in January 2013 showed metastases to liver. She was thenassessed as a good candidate for enrollment to IMMU-132 (hRS7-SN-38)investigational study. Comorbidities in her medical history includeasthma, diabetes mellitus, hypertension, hypercholesteremia, heartmurmur, hiatal hernia, hypothyroidism, carpel tunnel syndrome, glaucoma,depression, restless leg syndrome, and neuropathy. Her surgical historyincludes tubo-ligation (1975), thyroidectomy (1983), cholescystectomy(2001), carpel tunnel release (2008), and glaucoma surgery.

At the time of entry into this trial, her target lesion was a 3.1-cmtumor in the left lobe of the liver. Non-target lesions included severalhypo-attenuated masses in the liver. Her baseline CEA was 781 ng/m.

After the patient signed the informed consent, IMMU-132 was given on aonce-weekly schedule by infusion for 2 consecutive weeks, then a rest ofone week, this constituting a treatment cycle. These cycles wererepeated as tolerated. The first infusion of IMMU-132 (8 mg/kg) wasstarted on Feb. 15, 2013, and completed without notable events. Sheexperienced nausea (Grade 2) and fatigue (Grade 2) during the course ofthe first cycle and has been continuing the treatment since then withoutmajor adverse events. She reported alopecia and constipation in March2013. The first response assessment done (after 6 doses) on Apr. 8, 2013showed a shrinkage of target lesion by 29% by computed tomography (CT).Her CEA level decreased to 230 ng/ml on Mar. 25, 2013. In the secondresponse assessment (after 10 doses) on May 23, 2013, the target lesionshrank by 39%, thus constituting a partial response by RECIST criteria.She has been continuing treatment as of Jun. 14, 2013, receiving 6cycles constituting 12 doses of hRS7-SN-38 (IMMU-132) at 8 mg/kg. Heroverall health and clinical symptoms improved considerably sincestarting this investigational treatment.

Example 15 Use of hRS7-SN-38 (IMMU-132) to Treat Therapy-RefractiveMetastatic Breast Cancer

The patient was a 57-year-old woman with stage IV, triple-negative,breast cancer (ER/PR negative, HER-neu negative), originally diagnosedin 2005. She underwent a lumpectomy of her left breast in 2005, followedby Dose-Dense ACT in adjuvant setting in September 2005. She thenreceived radiation therapy, which was completed in November. Localrecurrence of the disease was identified when the patient palpated alump in the contralateral (right) breast in early 2012, and was thentreated with CMF (cyclophosphamide, methotrexate, 5-fluorouracil)chemotherapy. Her disease recurred in the same year, with metastaticlesions in the skin of the chest wall. She then received acarboplatin+TAXOL® chemotherapy regimen, during which thrombocytopeniaresulted. Her disease progressed and she was started on weeklydoxorubicin, which was continued for 6 doses. The skin disease also wasprogressing. An FDG-PET scan on Sep. 26, 2012 showed progression ofdisease on the chest wall and enlarged, solid, axillary nodes. Thepatient was given oxycodone for pain control.

She was given IXEMPRA® from October 2012 until February 2013 (every 2weeks for 4 months), when the chest wall lesion opened up and bled. Shewas then put on XELODA®, which was not tolerated well due to neuropathyin her hands and feet, as well as constipation. The skin lesions wereprogressive and then she was enrolled in the IMMU-132 trial after givinginformed consent. The patient also had a medical history ofhyperthyroidism and visual disturbances, with high risk of CNS disease(however, brain MM was negative for CNS disease). At the time ofenrollment to this trial, her cutaneous lesions (target) in the rightbreast measured 4.4 cm and 2.0 cm in the largest diameter. She hadanother non-target lesion in the right breast and one enlarged lymphnode each in the right and left axilla.

The first IMMU-132 infusion (12 mg/kg) was started on Mar. 12, 2013,which was tolerated well. Her second infusion was delayed due to Grade 3absolute neutrophil count (ANC) reduction (0.9) on the scheduled day ofinfusion, one week later. After a week delay and after receivingNEULASTA®, her second IMMU-132 was administered, with a 25% dosereduction at 9 mg/kg. Thereafter she has been receiving IMMU-132 onschedule as per protocol, once weekly for 2 weeks, then one week off.Her first response assessment on May 17, 2013, after 3 therapy cycles,showed a 43% decrease in the sum of the long diameter of the targetlesions, constituting a partial response by RECIST criteria. She iscontinuing treatment at the 9 mg/kg dose level. Her overall health andclinical symptoms improved considerably since she started treatment withIMMU-132.

Example 16 Use of hRS7-SN-38 (IMMU-132) to Treat Refractory, Metastatic,Non-Small Cell Lung Cancer

This is a 60-year-old man diagnosed with non-small cell lung cancer. Thepatient is given chemotherapy regimens of carboplatin, bevacizumab for 6months and shows a response, and then after progressing, receivesfurther courses of chemotherapy with carboplatin, etoposide, TAXOTERE®,gemcitabine over the next 2 years, with occasional responses lasting nomore than 2 months. The patient then presents with a left mediastinalmass measuring 6.5×4 cm and pleural effusion.

After signing informed consent, the patient is given IMMU-132 at a doseof 18 mg/kg every other week. During the first two injections, briefperiods of neutropenia and diarrhea are experienced, with 4 bowelmovements within 4 hours, but these resolve or respond to symptomaticmedications within 2 days. After a total of 6 infusions of IMMU-132, CTevaluation of the index lesion shows a 22% reduction, just below apartial response but definite tumor shrinkage. The patient continueswith this therapy for another two months, when a partial response of 45%tumor shrinkage of the sum of the diameters of the index lesion is notedby CT, thus constituting a partial response by RECIST criteria.

Example 17 Use of hRS7-SN-38 (IMMU-132) to Treat Refractory, Metastatic,Small-Cell Lung Cancer

This is a 65-year-old woman with a diagnosis of small-cell lung cancer,involving her left lung, mediastinal lymph nodes, and MRI evidence of ametastasis to the left parietal brain lobe. Prior chemotherapy includescarboplatin, etoposide, and topotecan, but with no response noted.Radiation therapy also fails to control her disease. She is then givenIMMU-132 at a dose of 18 mg/kg once every three weeks for a total of 5infusions. After the second dose, she experiences hypotension and aGrade 2 neutropenia, which improve before the next infusion. After thefifth infusion, a CT study shows 13% shrinkage of her target left lungmass. MRI of the brain also shows a 10% reduction of this metastasis.She continues her IMMU-132 dosing every 3 weeks for another 3 months,and continues to show objective and subjective improvement of hercondition, with a 25% reduction of the left lung mass and a 21%reduction of the brain metastasis.

Example 18 Therapy of a Gastric Cancer Patient with Stage IV MetastaticDisease with hRS7-SN-38 (IMMU-132)

This patient is a 60-year-old male with a history of smoking and periodsof excessive alcohol intake over a 40-year-period. He experiences weightloss, eating discomfort and pain not relieved by antacids, frequentabdominal pain, lower back pain, and most recently palpable nodes inboth axilla. He seeks medical advice, and after a workup is shown tohave an adenocarcinoma, including some squamous features, at thegastro-esophageal junction, based on biopsy via a gastroscope.Radiological studies (CT and FDG-PET) also reveal metastatic disease inthe right and left axilla, mediastinal region, lumbar spine, and liver(2 tumors in the right lobe and 1 in the left, all measuring between 2and 4 cm in diameter). His gastric tumor is resected and he is then puton a course of chemotherapy with epirubicin, cisplatin, and5-fluorouracil. After 4 months and a rest period of 6 weeks, he isswitched to docetaxel chemotherapy, which also fails to control hisdisease, based on progression confirmed by CT measurements of themetastatic tumors and some general deterioration.

The patient is then given therapy with IMMU-132 (hRS7-SN-38) at a doseof 10 mg/kg infused every-other-week for a total of 6 doses, after whichCT studies are done to assess status of his disease. These infusions aretolerated well, with some mild nausea and diarrhea, contolled withsymptomatic medications. The CT studies reveal that the sum of his indexmetastatic lesions has decreased by 28%, so he continues on this therapyfor another 5 courses. Follow-up CT studies show that the diseaseremains about 35% reduced by RECIST criteria from his baselinemeasurements prior to IMMU-132 therapy, and his general condition alsoappears to have improved, with the patient regaining an optimisticattitude toward his disease being under control.

Example 19 Therapy of Advanced Colon Cancer Patient Refractory to PriorChemo-Immunotherapy, Using Only IMMU-130 (Labetuzumab-SN-38)

The patient is a 50-year-old man with a history of stage-IV metastaticcolonic cancer, first diagnosed in 2008 and given a colectomy andpartial hepatectomy for the primary and metastatic colonic cancers,respectively. He then received chemotherapy, as indicated FIG. 8, whichincluded irinotecan, oxaliplatin, FOLFIRINOX (5-fluoruracil, leucovorin,irinotecan, oxaliplatin), and bevacizumab, as well as bevacizumabcombined with 5-fluorouracil/leucovorin, for almost 2 years. Thereafter,he was given courses of cetuximab, either alone or combined with FOLFIRI(leucovorin, 5-flurouracil, irinotecan) chemotherapy during the nextyear or more. In 2009, he received radiofrequency ablation therapy tohis liver metastasis while under chemo-immunotherapy, and in late 2010he underwent a wedge resection of his lung metastases, which wasrepeated a few months later, in early 2011. Despite havingchemo-immunotherapy in 2011, new lung metastases appeared at the end of2011, and in 2012, both lung and liver metastases were visualized. Hisbaseline plasma carcinoembryonic antigen (CEA) titer was 12.5 ng/mL justbefore undergoing the antibody-drug therapy with IMMU-130. The indexlesions chosen by the radiologist for measuring tumor size change bycomputed tomography were the mid-lobe of the right lung and the livermetastases, both totaling 91 mm as the sum of their longest diameters atthe baseline prior to IMMU-130 (anti-CEACAM5-SN-38) therapy.

This patient received doses of 16 mg/kg of IMMU-130 by slow IV infusionevery other week for a total of 17 treatment doses. The patienttolerated the therapy well, having only a grade 1 nausea, diarrhea andfatigue after the first treatment, which occurred after treatments 4 and5, but not thereafter, because he received medication for theseside-effects. After treatment 3, he did show alopecia (grade 2), whichwas present during the subsequent therapy. The nausea, diarrhea, andoccasional vomiting lasted only 2-3 days, and his fatigue after thefirst infusion lasted 2 weeks. Otherwise, the patient tolerated thetherapy well. Because of the long duration of receiving this humanized(CDR-grafted) antibody conjugated with SN-38, his blood was measured foranti-labetuzumab antibody, and none was detected, even after 16 doses.

The first computed tomography (CT) measurements were made after 4treatments, and showed a 28.6% change from the sum of the measurementsmade at baseline, prior to this therapy, in the index lesions. After 8treatments, this reduction became 40.6%, thus constituting a partialremission according to RECIST criteria. This response was maintained foranother 2 months, when his CT measurements indicated that the indexlesions were 31.9% less than the baseline measurements, but somewhathigher than the previous decrease of 40.6% measured. Thus, based oncareful CT measurements of the index lesions in the lung and liver, thispatient, who had failed prior chemotherapy and immunotherapy, includingirinotecan (parent molecule of SN-38), showed an objective response tothe active metabolite of irintotecan (or camptotechin), SN-38, whentargeted via the anti-CEACAM5 humanized antibody, labetuzumab (hMN-14).It was surprising that although irinotecan (CPT-11) acts by releasingSN-38 in vivo, the SN-38 conjugated anti-CEACAM5 antibody provedeffective in a colorectal cancer patient by inducing a partial responseafter the patient earlier failed to respond to his lastirinotecan-containing therapy. The patient's plasma CEA titer reductionalso corroborated the CT findings: it fell from the baseline level of12.6 ng/mL to 2.1 ng/mL after the third therapy dose, and was between1.7 and 3.6 ng/mL between doses 8 and 12. The normal plasma titer of CEAis usually considered to be between 2.5 and 5.0 ng/mL, so this therapyeffected a normalization of his CEA titer in the blood.

Example 20 Therapy of a Patient with Advanced Colonic Cancer withIMMU-130

This patient is a 75-year-old woman initially diagnosed with metastaticcolonic cancer (Stage IV). She has a right partial hemicolectomy andresection of her small intestine and then receives FOLFOX,FOLFOX+bevacizumab, FOLFIRI+ramucirumab, and FOLFIRI+cetuximab therapiesfor a year and a half, when she shows progression of disease, withspread of disease to the posterior cul-de-sac, omentum, with ascites inher pelvis and a pleural effusion on the right side of her chest cavity.Her baseline CEA titer just before this therapy is 15 ng/mL. She isgiven 6 mg/kg IMMU-130 (anti-CEACAM5-SN-38) twice weekly for 2consecutive weeks, and then one week rest (3-week cycle), for more than20 doses, which is tolerated very well, without any major hematologicalor non-hematological toxicities. Within 2 months of therapy, her plasmaCEA titer shrinks modestly to 1.3 ng/mL, but at the 8-week evaluationshe shows a 21% shrinkage of the index tumor lesions, which increases toa 27% shrinkage at 13 weeks. Surprisingly, the patient's ascites andpleural effusion both decrease (with the latter disappearing) at thistime, thus improving the patient's overall status remarkably. Thepatient continues her investigational therapy.

Example 21 Gastric Cancer Patient with Stage IV Metastatic DiseaseTreated with IMMU-130

The patient is a 52-year-old male who sought medical attention becauseof gastric discomfort and pain related to eating for about 6 years, andwith weight loss during the past 12 months. Palpation of the stomacharea reveals a firm lump which is then gastroscoped, revealing anulcerous mass at the lower part of his stomach. This is biopsied anddiagnosed as a gastric adenocarcinoma. Laboratory testing reveals nospecific abnormal changes, except that liver function tests, LDH, andCEA are elevated, the latter being 10.2 ng/mL. The patent then undergoesa total-body PET scan, which discloses, in addition to the gastrictumor, metastatic disease in the left axilla and in the right lobe ofthe liver (2 small metastases). The patient has his gastric tumorresected, and then has baseline CT measurements of his metastatictumors. Four weeks after surgery, he receives 3 courses of combinationchemotherapy consisting of a regimen of cisplatin and 5-fluorouracil(CF), but does not tolerate this well, so is switched to treatment withdocetaxel. It appears that the disease is stabilized for about 4 months,based on CT scans, but then the patient's complaints of further weightloss, abdominal pain, loss of appetite, and extreme fatigue causerepeated CT studies, which show increase in size of the metastases by asum of 20% and a suspicious lesion at the site of the original gastricresection.

The patient is then given experimental therapy with IMMU-130(anti-CEACAM5-SN-38) on a weekly schedule of 8 mg/kg. He tolerates thiswell, but after 3 weeks shows a grade 2 neutropenia and grade 1diarrhea. His fourth infusion is postponed by one week, and then theweekly infusions are reinstituted, with no evidence of diarrhea orneutropenia for the next 4 injection. The patient then undergoes a CTstudy to measure his metastatic tumor sizes and to view the originalarea of gastric resection. The radiologist measures, according to RECISTcriteria, a decrease of the sum of the metastatic lesions, compared tobaseline prior to IMMU-130 therapy, of 23%. There does not seem to beany clear lesion in the area of the original gastric resection. Thepatient's CEA titer at this time is 7.2 ng/mL, which is much reducedfrom the pre-IMMU-130 baseline value of 14.5 ng/mL. The patientcontinues on weekly IMMU-130 therapy at the same dose of 8.0 mg/kg, andafter a total of 13 infusions, his CT studies show that one livermetastasis has disappeared and the sum of all metastatic lesions isdecreased by 41%, constituting a partial response by RECIST. Thepatient's general condition improves and he resumes his usual activitieswhile continuing to receive a maintenance therapy of 8 mg/kg IMMU-130every third week for another 4 injections. At the last measurement ofblood CEA, the value is 4.8 ng/mL, which is within the normal range fora smoker, which is the case for this patient.

Example 22 Therapy of Relapsed Triple-Negative Metastatic Breast Cancerwith hMN-15-SN-38

A 58-year-old woman with triple-negative metastatic breast cancerformerly treated with bevacizumab plus paclitaxel, without response,presents with metastases to several ribs, lumbar vertebrae, a solitarylesion measuring 3 cm in diameter in her left lung, with considerablebone pain and fatigue. She is given an experimental therapy with theanti-CEACAM6 humanized monoclonal antibody, hMN-15 IgG, conjugated with6 molecules of SN-38 per IgG. She is given an infusion of 12 mg/kg everythird week, repeated for 4 doses, as a course of therapy. Except fortransient grade 2 neutropenia and some initial diarrhea, she toleratesthe therapy well, which is then repeated, after a rest of 2 months, foranother course. Radiological examination indicates that she has partialresponse by RECIST criteria, because the sum of the diameters of theindex lesions decrease by 39%. Her general condition, including bonepain, also improves, and she returns to almost the same level ofactivity as prior to her illness.

Example 23 Therapy of Relapsed, Generally Refractive, Metastatic ColonicCarcinoma with hMN-15-SN-38

A 46-year-old woman has Stage IV metastatic colonic cancer, with a priorhistory of resection of the primary lesion that also had synchronousliver metastases to both lobes of the liver, as well as a single focusof spread to the right lung; these metastases measured, by CT, between 2and 5 cm in diameter. She undergoes various courses of chemotherapy overa period of 3 years, including 5-fluorouracil, leucovorin, irinotecan,oxaliplatin, cetuximab, and bevacizumab. On two occasions, there isevidence of stabilization of disease or a short-term response, but noreduction of 30% or more of her measured lesions. Her plasma CEA titerat baseline prior to hMN-14-SN-38 therapy is 46 ng/mL, and her totalindex lesions measure a sum of 92 mm.

Therapy with hMN-15-SN-38 is instituted at 12 mg/kg weekly for 2 weeks,with a rest period of one week thereafter, within a 21-day cycle. Thiscycle is repeated 3 times, with only transient neutropenia andgastrointestinal side effects (nausea, vomiting, diarrhea).Surprisingly, despite failing to respond to FOLFIRI therapy (whichincludes irinotecan, or CPT-11), the patient shows a partial response byRECIST criteria after completing her therapy. She is then placed on amaintenance schedule of this therapy at a dose of 16 mg/kg once everymonth for the next 6 months. Followup scans show that her diseaseremains under control as a partial response (PR), and the patient isgenerally in good condition with a 90% Kaaarnofsky performance status.

Example 24 Colonic Cancer Patient with Stage IV Metastatic DiseaseTreated with Anti-CSAp-SN-38 Conjugate

This patient presents with colonic cancer metastases to the left lobe ofthe liver and to both lungs, after having a resection of a 9-cm sigmoidcolon adenocarinoma, followed by chemo-immunotherapy with FOLIFIRI andcetuximab for 6 months, and then FOLFOX followed by bevacizumab for anadditional period of about 9 months. Ten months after the initialresection and then commencement of therapy, the stable disease thoughtto be present shows progression by the lesions growing and a newmetastasis appearing in the left adrenal gland. Her plasma CEA at thistime is 52 ng/mL, and her general condition appears to havedeteriorated, with abdominal pains, fatigue, and borderline anemia,suggesting possibly internal bleeding.

She is now given an SN-38 conjugate of hMu-9 (anti-CSAp) antibody at adose of 12 mg/kg weekly for two weeks, with one week rest, as atreatment cycle, and then repeated for additional treatment cycles,measuring her blood counts every week and receiving atropine medicationto gastrointestinal reactions. Grade 2 alopecia is noted after the firsttreatment cycle, but only a Grade 1 neutropenia. After 3 treatmentcycles, her plasma CEA titer is reduced to 19 ng/ml, and at this timeher CT measurements show a decrease of the index lesions in the liverand lungs by 24.1%. After an additional 3 courses of therapy, she showsa CT reduction of the index lesions of 31.4%, and a decrease in the sizeof the adrenal mass by about 40%. This patient is considered to beresponding to anti-CSAp-SN-38 antibody-drug therapy, and continues onthis therapy. Her general condition appears to be improved, with lessfatigue, no abdominal pain or discomfort, and generally more energy.

Example 25 Treatment of Breast Cancer with Anti-CEACAM6-SN-38Immunoconjugate

This patient has triple-negative (does not express estrogen receptor,progesterone receptor or Her2/neu) metastatic breast cancer that isrelapsed after several different therapies over the past 3 years. Shepresents with several small tumors in both lungs, as well as metastasesto her C4, C5, T2, and T3 vertebrae, as well as the several ribsbilaterally. She is under standard therapy for her osteolytic lesions,and now begins treatment with hMN-15-SN-38 at a dose of 16 mg/kgonce-weekly for 3 weeks, with a pause of one week, and then resumes this3-weekly-cycle therapy two more times. At 2 weeks post therapy, CT scansare performed to evaluate response, and it is noted that 2 of the smalllung metastases have disappeared while 1 of the larger lesions appearsto have diminished by about 40%. The metastases to the vertebrae arestill present, but the C4 and C5 lesions appear smaller by about 25%. Ofthe metastases to the ribs, 2 of 6 small lesions appear to be very muchdiminished in size, and are not certain as to being viable or smallareas of scar or necrosis. The patient's tumor markers, as well as herLDH titers, appear to show either stable or reduced levels, indicatingthat disease progression has been halted and there is also some evidenceof disease reduction. Subjectively, the patient is feeling much better,with less fatigue and bone pain and improved breathing. She hasexperienced some minimal nausea and vomiting after each therapy, whichresolved within a week. The only other side effect has been a transientthrombocytopenia, which also resolves within 7 days. She is beingobserved and will resume therapy cycles within 2 months.

Example 26 Treatment of Metastatic Colon Cancer with CombinationAnti-CEACAM5 and Anti-CEACAM6-SN-38 Immunoconjugates

This patient has metastatic colonic cancer, with CT evidence of diseasein the liver (5 cm lesion in right lobe and 3 cm lesion in left lobe),as well as 2 metastases (2- and 3-cm sizes) to the right lung. Theprimary cancer of the colon was previously resected and the patient hadcourses of post-operative therapy because of metachronous metastases tothe liver and lungs. During therapy, the liver metastases grow and theone lung metastasis becomes two, so the patient is a candidate forexperimental chemoimmunotherapy. He is then begun on a course of doubleantibody-drug conjugates, labetuzumab (hMN-14)-SN-38 and hMN-15-SN-38,each given on alternate days at doses of 8 mg/kg, once weekly for 2weeks, and then repeated monthly for 4 months. Two weeks after therapy,the patient's status is evaluated by CT and lab tests. The CT scansreveal that the large tumor in the right liver lobe is reduced by 50%,the tumor in the left lobe by about 33%, and the lung metastases byabout 20% cumulatively for both tumors. His blood CEA titer isdiminished from 22 ng/mL at onset of therapy to 6 ng/mL at thisfollowup. Subjectively, the patient states he is feeling stronger andalso appears to have more vigor in daily activities. Side effects aretransient thrombocytopenia and leucopenia, returning to normal rangeswithin 2 weeks after therapy, and several bouts of nausea and vomiting,controlled by anti-emetic medication. It is planned that the patientwill resume these therapy cycles in about 2 months, following anotherworkup for disease status.

Example 27 Continuous Infusion of Antibody-Drug Conjugates

The patient was previously resected for a rectal carcinoma and receivespre- and post-operative radiochemotherapy as per conventional treatment.She has been free of tumor for four years, but now presents with 3 smallmetastatic lesions to the right liver lobe, discovered by routine CT andfollowup blood CEA values, which rise to 6.3 ng/mL from the 3.0 ng/mLpost initial therapy. She is given an indwelling catheter and acontinuous infusion of labetuzumab-SN-28 at a dose of 2 mg/kg over 17days. She then receives a repeat continuous infusion therapy 5 weekslater, now for 3 weeks, at 1 mg/kg. Three weeks later, CT scans andblood CEA monitoring reveal that 1 of the liver metastases hasdisappeared and the other two are the same or slightly smaller. Theblood CEA titer now measures 2.4 ng/mL. She is not symptomatic, and onlyexperiences grade 2 nausea and vomiting while under therapy, and grade 2neutropenia, both resolving with time.

Example 28 Therapy of Advanced Metastatic Colon Cancer with Anti-CEACAM5Immunoconjugate

The patient is a 50-year-old male who fails prior therapies formetastatic colon cancer. The first line of therapy isFOLFIRINOX+AVASTIN® (built up in a stepwise manner) starting with IROX(Irinotecan+Oxaliplatin) in the first cycle. After initiating thistreatment the patient has a CT that shows decrease in the size of livermetastases. This is followed by surgery to remove tumor tissue. Adjuvantchemotherapy is a continuation of the first line regimen (without theIROX part) that resulted in a transient recurrence-free period. Afterabout a 1 year interval, a CT reveals the recurrence of livermetastases. This leads to the initiation of the second line regimen(FOLFIRI+Cetuximab). Another CT shows a response in liver metastases.Then RF ablation of liver metastases is performed, followed bycontinuation of adjuvant chemotherapy with FOLFIRINOX+Cetuximab,followed by maintenance Cetuximab for approximately one year. Another CTscan shows no evidence of disease. A further scan shows possible lungnodules, which is confirmed. This leads to a wedge resection of the lungnodules. Subsequently FOLFIRI+Cetuximab is restarted and continued.Later CT scans show both lung and liver metastases.

At the time of administration of the hMN-14-SN-38 immunoconjugate, thepatient has advanced metastatic colon cancer, with metastases of bothlung and liver, which is unresponsive to irinotecan (camptothecin). ThehMN-14-SN-38 immunoconjugate is administered at a dosage of 12 mg/kg,which is repeated every other week. The patient shows a partial responsewith reduction of metastatic tumors by RECIST criteria.

Of note is that only one patient in this 12 mg/kg (given every otherweek) cohort shows a grade 2 hematological (neutropenia) and mostpatients have grade 1 or 2 nausea, vomiting, or alopecia—which are signsof activity of the antibody-drug conjugate, but well tolerated. Theeffect of the antibody moiety in improved targeting of the camptothecinaccounts for the efficacy of the SN-38 moiety in the cancer that hadbeen previously resistant to unconjugated irinotecan.

Example 29 Treatment of Metastatic Pancreatic Cancer withAnti-MUC5ac-SN-38 Immunoconjugate

This 44-year-old patient has a history of metastatic pancreaticcarcinoma, with inoperable pancreas ductal adenocarcinoma in thepancreas head, and showing metastases to left and right lobes of theliver, the former measuring 3×4 cm and the latter measuring 2×3 cm. Thepatient is given a course of gemcitabine but shows no objectiveresponse. Four weeks later, he is given hPAM4-SN-38 i.v. at a dose of 8mg/kg twice-weekly for 2 weeks, with one week off, and then repeated foranother 2 cycles. CT studies are done one week later and show a totalreduction in tumor mass (all sites) of 32% (partial response), alongsidea drop in his blood CA19-9 titer from 220 at baseline to 75 at the timeof radiological evaluation. The patient shows only grade 1 nausea andvomiting after each treatment with the antibody-drug conjugate, and agrade 2 neutropenia at the end of the last treatment cycle, whichresolves 4 weeks later. No premedication for preventing infusionreactions is given.

Example 30 Use of hL243-SN-38 to Treat Therapy-Refractive MetastaticColonic Cancer (mCRC)

The patient is a 67-year-old man who presents with metastatic coloncancer. Following transverse colectomy shortly after diagnosis, thepatient then receives 4 cycles of FOLFOX chemotherapy in a neoadjuvantsetting prior to partial hepatectomy for removal of metastatic lesionsin the left lobe of the liver. This is followed by an adjuvant FOLFOXregimen for a total of 10 cycles of FOLFOX.

CT shows metastases to liver. His target lesion is a 3.0-cm tumor in theleft lobe of the liver. Non-target lesions included severalhypo-attenuated masses in the liver. Baseline CEA is 685 ng/mL.

After the patient signs the informed consent, hL243-SN-38 (10 mg/kg) isgiven every other week for 4 months. The patient experiences nausea(Grade 2) and fatigue (Grade 2) following the first treatment andcontinues the treatment without major adverse events. The first responseassessment done (after 8 doses) shows shrinkage of the target lesion by26% by computed tomography (CT) and his CEA level decreases to 245ng/mL. In the second response assessment (after 12 doses), the targetlesion has shrunk by 35%. His overall health and clinical symptoms areconsiderably improved.

Example 31 Treatment of Relapsed Follicular Lymphoma with IMMU-114-SN-38(anti-HLA-DR-SN-38)

After receiving R-CHOP chemotherapy for follicular lymphoma presentingwith extensive disease in various regional lymph nodes (cervical,axillary, mediastinal, inguinal, abdominal) and marrow involvement, this68-year-old man is given the experimental agent, IMMU-114-SN-38(anti-HLA-DR-SN-38) at a dose of 10 mg/kg weekly for 3 weeks, with arest of 3 weeks, and then a second course for another 3 weeks. He isthen evaluated for change in index tumor lesions by CT, and shows a 23%reduction according CHESON criteria. The therapy is repeated for another2 courses, which then shows a 55% reduction of tumor by CT, which is apartial response.

Example 32 Treatment of Relapsed Chronic Lymphocytic Leukemia withIMMU-114-SN-38

A 67-year-old man with a history of CLL, as defined by the InternationalWorkshop on Chronic Lymphocytic Leukemia and World Health Organizationclassifications, presents with relapsed disease after prior therapieswith fludarabine, dexamethasone, and rituximab, as well as a regimen ofCVP. He now has fever and night sweats associated with generalized lymphnode enlargement, a reduced hemoglobin and platelet production, as wellas a rapidly rising leukocyte count. His LDH is elevated and thebeta-2-microglobulin is almost twice normal. The patient is giventherapy with IMMU-114-SN-38 conjugate at a dosing scheme of 8 mg/kgweekly for 4 weeks, a rest of 2 weeks, and then the cycle repeatedagain. Evaluation shows that the patient's hematological parameters areimproving and his circulating CLL cells appear to be decreasing innumber. The therapy is then resumed for another 3 cycles, after whichhis hematological and lab values indicate that he has a partialresponse.

Example 33 Use of hMN-15-SN-38 to Treat Refractory, Metastatic,Non-Small Cell Lung Cancer

The patient is a 58-year-old man diagnosed with non-small cell lungcancer. He is initially given chemotherapy regimens of carboplatin,bevacizumab for 6 months and shows a response, and then afterprogressing, receives further courses of chemotherapy with carboplatin,etoposide, TAXOTERE®, gemcitabine over the next 2 years, with occasionalresponses lasting no more than 2 months. The patient then presents witha left mediastinal mass measuring 5.5 x 3.5 cm and pleural effusion.

After signing informed consent, the patient is given hMN-15-SN-38 at adose of 12 mg/kg every other week. During the first two injections,brief periods of neutropenia and diarrhea are experienced, but theseresolve or respond to symptomatic medications within 2 days. After atotal of 6 infusions of hMN-15-SN-38, CT evaluation of the target lesionshows a 22% reduction. The patient continues with this therapy foranother two months, when a partial response of 45% is noted by CT.

Example 34 Treatment of Follicular Lymphoma Patient with hA19-SN-38

A 60-year-old male presents with abdominal pain and the presence of apalpable mass. The patient has CT and FDG-PET studies confirming thepresence of the mass with pathologic adenopathies in the mediastinum,axillary, and neck nodes. Lab tests are unremarkable except for elevatedLDH and beta-2-microglobulin. Bone marrow biopsy discloses severalparatrabecular and perivascular lymphoid aggregates. These arelymphocytic with expression of CD20, CD19, and CD10 by immunostaining.The final diagnosis is grade-2 follicular lymphoma, stage IVA, with aFLIPI score of 4. The longest diameter of the largest involved node is 7cm. The patient is given a humanized anti-CD19 monoclonal antibody IgG(hA19) conjugated with SN-38 (6 drug molecules per IgG). The dosing is 6mg/kg weekly for 4 consecutive weeks, two weeks off, and then repeatedcycles of 4 treatment weeks for every 6 weeks. After 5 cycles, bonemarrow and imaging (CT) evaluations show a partial response, where themeasurable lesions decrease by about 60% and the bone marrow is muchless infiltrated. Also, LDH and beta-2-microglobulin titers alsodecrease.

Example 35 Treatment of Relapsed Precursor B-Cell ALL with hA19-SN-38

This 51-year-old woman has been under therapy for precursor,Philadelphia chromosome-negative, B-cell ALL, which shows the ALL cellsstain for CD19, CD20, CD10, CD38, and CD45. More than 20% of the marrowand blood lymphoblasts express CD19 and CD20. The patient has receivedprior therapy with clofarabine and cytarabine, resulting in considerablehematological toxicity, but no response. A course of high-dosecytarabine (ara-C) was also started, but could not be tolerated by thepatient. She is given hA19-SN-38 therapy at weekly doses by infusion of6 mg/kg for 5 weeks, and then a 2-week rest, with repetition of thistherapy two more times. Surprisingly, she shows improvement in her bloodand marrow counts, sufficient for a partial response to be determined.After a rest of 2 months because of neutropenia (grade 3), therapyresumes at 8 mg/kg every other week for another 4 courses. At this time,she is much improved and is under consideration for maintenance therapyto try to bring her to a stage where she could be a candidate forstem-cell transplantation.

Example 36 Treatment of Lymphoma with Anti-CD22-SN-38 Immunoconjugate

The patient is a 62 year-old male with relapsed diffuse large B-celllymphoma (DLBCL). After 6 courses of R-CHOP chemoimmunotherapy, he nowpresents with extensive lymph node spread in the mediastinum, axillary,and inguinal lymph nodes. He is given epratuzumab-SN-38 (anti-CD22) at adose of 12 mg/kg weekly×3, with one week off, and then repeated againfor another two cycles. One week later, the patient is evaluated by CTimaging, and his total tumor bulk is measured and shows a decrease of35% (partial response), which appears to be maintained over the next 3months. Side effects are only thrombocytopenia and grade 1 nausea andvomiting after therapy, which resolve within 2 weeks. No pretherapy forreducing infusion reactions is given.

Example 37 Combination Therapy of Follicular Lymphoma with Veltuzumaband Epratuzumab-SN-38 in the Frontline Setting

A 35-year-old woman is diagnosed with a low-grade and good FLIPI scorefollicular lymphoma, presenting in her cervical lymph nodes, bothaxilla, and mediastinum. Her spleen is not enlarged, and bone marrowbiopsy does not disclose disease involvement. She is symptomatically notmuch affected, with only periods of elevated temperature, night sweats,and somewhat more fatigued than usual. Her physician decides not toundertake a watch-and-wait process, but to give this woman aless-aggressive therapy combining a subcutaneous course of the humanizedanti-CD20 monoclonal antibody, veltuzumab, weekly×4 weeks (200 mg/m²)combined with two weekly courses of the anti-CD22 epratuzumab-SN-38,each infusion being a dose of 8 mg/kg. This combination therapy isrepeated 2 months later, and after this the patient is evaluated by CTand FDG-PET imaging studies, as well as a bone marrow biopsy.Surprisingly, about a 90% reduction of all disease is noted, and shethen is given another course of this combination therapy after a rest of4 weeks. Evaluation 4 weeks later shows a radiological (and bone marrowbiopsy) complete response. Her physician decides to repeat this courseof therapy 8 months later, and radiological/pathological tests show asustained complete remission.

Example 38 Frontline Therapy of Follicular Lymphoma UsingVeltuzumab-SN-38

The patient is a 41-year-old woman presenting with low-grade follicularlymphoma, with measurable bilateral cervical and axillary lymph nodes(2-3 cm each), mediastinal mass of 4 cm diameter, and an enlargedspleen. She is given 3 courses of veltuzumab-SN-38 (anti-CD20-SN-38)therapy, with each course consisting of 10 mg/kg infused once every 3weeks. After completion of therapy, her tumor measurements by CT show areduction of 80%. She is then given 2 additional courses of therapy, andCT measurements indicate that a complete response is achieved. This isconfirmed by FDG-PET imaging.

Example 39 Therapy of Relapsed DLBCL with 1F5 Humanized AntibodyConjugated with SN-38

A 53-year-old woman presents with recurrent diffuse large B-celllymphoma at mediastinal and abdominal para-aortic sites 8 months aftershowing a partial response to R-CHOP chemotherapy given for 6 cycles.She refuses to have more cytotoxic chemotherapy, so is given a mildertherapy consisting of 10 mg/kg humanized 1F5 anti-CD20 monoclonalantibody, conjugated to about 6 molecules of SN-28 per molecule ofantibody, once weekly every other week for 5 infusions. CT and FDG-PETstudies indicate a further reduction of her lymphomas by 40%, so after arest period of 4 weeks, therapy is resumed at a dose of 8 mg/kg every 3weeks for a total of 5 infusions. Evaluation of her disease reveals areduction of about 80%.

Example 40 Therapy of Relapsed Chronic Lymphocytic Leukemia withRituximab-SN-38

A 62-year-old man with an 8-year history of CLL, having responded in thepast to fludarabine, cyclophosphamide, and rituximab therapy, and afterrelapse to ibrutinib for a partial response lasting 9 months, presentswith progressing disease. The patient is given rituximab-SN-38monotherapy at a schedule of 12 mg/kg every 2 weeks for 3 courses,reduced to 8 mg/kg every other week for another 4 courses. Sustainedimprovement in cytopenias, reflected by more than 50% or a hemoglobinlevel higher than 11 g per deciliter, an absolute neutrophil counthigher than 1500 cells per cmm, or a platelet count higher than 11 k/cmmis observed, which was durable for about 9 months.

Example 41 Frontline Therapy of DLBCL with Veltuzumab-SN-38 Combinedwith Bendamustine

A 59-year-old man presents with multiple sites of DLBCL, includingchest, abdominal, inguinal lymph nodes, and enlarged spleen, asconfirmed by CT, FDG-PET, and immunohistological/pathological diagnoses.Bendamustine is given at a dose of 90 mg/m² on days 1 and 2, combinedwith veltuzumab-SN-38 at a dose of 6 mg/kg on days 7 and 14, given every4 weeks for four cycles. Evaluation radiologically thereafter shows apartial response. After a rest of 2 months, the therapy is repeated foranother 2 cycles, and radiological assessment then shows a completeresponse. Cytopenias, mostly neutropenia, is manageable and does notachieve a grade 3 level.

Example 42 Frontline Therapy of Mantle Cell Lymphoma (MCL) withVeltuzumab-SN-38 Combined with Lenalidomide

The patient is a 68-year-old man diagnosed with MCL after presentingwith a GI complaint and lethargy. Colonoscopy discloses a 7-cm cecalmass, and his workup reveals that he has Stage IV disease. He is given acombination therapy of lenalidomide, 25 mg orally daily on days 1 to 21every 28 days. After two cycles, he is given veltuzumab-SN-38 everyother week at a dose of 10 mg/kg for 3 treatments, with a 2-week rest.This is then repeated again. Two weeks after completion of this therapy,the patient shows a partial response of his measured index lesion andreduction of other lymph nodes visualized. Four months later,lenalidomide therapy is repeated for 21 days, followed by 2 courses ofveltuzumab-SN-38. His disease is then shown to be reduced even further,although not yet a complete response.

Example 43 Epratuzumab-SN-38 Therapy of a Patient withRelapsed/Refractory Diffuse Large B-Cell Lymphoma (DLBCL)

A 65-year-old man with symptoms of weight loss undergoes a biopsy of anepigastric mass, which is diagnosed as a diffuse large B-cell lymphoma.He is treated with 6 cycles of standard R-CHOP (rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone). He hasprolonged and persistent neutropenia (700 ANC) with mildthrombocytopenia (50-70 k/mL), but no real anemia. His IPI is high. Theepigastric mass does not show any change following this therapy, and heis put on a mild treatment regimen of anti-CD22 epratuzumab-SN-38. Thisis a regimen of 4 mg/kg infused every other week for 4 infusions, thenonce every third week for another 3 infusions. His epigastric lymphomais measured by CT one week later and shows a marked reduction by 52%.The patient is continues this treatment every third week for another 3months, and continues to show this reduction of his lymphoma mass withstabilization of his weight and improvement of his energy andactivities.

Example 44 Humanized RFB4 Therapy of a Patient with Relapsed FollicularLymphoma

A 42-year-old woman presents with a sharp, constant and severe pain inher lower abdomen which radiates to her back. Laboratory tests areunremarkable, but an abdominal ultrasound shows a heterogeneous solidmass in the anterior lower left, measuring 7.5×6.2×7.0 cm. CT scansreveal a large mass within the left small-bowel mesentery, withinvolvement of adjacent lymph nodes. A CT-guided needle biopsy of themass shows that it is a follicular lymphoma, grade 3.Immunohistochemistry shows a B-cell type with positive results for CD19,CD20, CD22, Bcl-2 and Bcl-6. PET studies reveal no disease above thediaphragm, in the bone marrow, or in the spleen, but bone marrow biopsydoes confirm involvement of lymphoma. The patient undergoes 6 cycles ofR-CHOP chemotherapy, resulting in a complete response to this therapy 4months later. However, 10 months later, she undergoes a relapse, withrecurrence of her abdominal mass and adjacent lymph nodes, as well as anenlarged spleen and more bone marrow involvement as determined by PETand biopsy studies. She now begins a course of therapy with thehumanized anti-CD22 monoclonal antibody, RFB4, conjugated with 6 SN-38molecules per IgG, at a dose of 8 mg/kg weekly for 3 weeks, and thencontinued at 8 mg/kg every other week for another 4 treatments. Twoweeks later, she undergoes CT and FDG-PET studies, and her abdominallesions and spleen show a reduction of 40%, and a general decrease ofbone marrow involvement. After a rest of 4 weeks, therapy at 4 mg/kgweekly for 4 weeks, followed by 6 mg/kg every other week for another 5treatments are implemented, with a further measurable reduction of thesum of the sizes of all measured lesions by a total of 60%. The patientcontinues on a maintenance therapy of hRFB4-SN-38 of 8 mg/kg oncemonthly for the next 5 months, and maintains her therapeutic response.

Example 45 Epratuzumab-SN-38 Therapy of a Patient withRelapsed/Refractory Acute Lymphoblastic Leukemia

A 29-year-old male with CD22+ precursor B-cell acute lymphoblasticleukemia (ALL) has not responded to therapy with PEG-asparaginase,cyclophosphamide, daunorubicin, cytarabine (ara-C), vincristine,leucovorin, prednisone, methotrexate, and 6-mercaptopurine, andsupportive therapy with G-CSF (Neupogen), given as induction/maintenancetherapies under a modified Larson protocol. The patient's leukemia isPhiladelphia chromosome-negative. Based on blood and marrow leukemiablast counts, the patient shows only a minimal response, with diseaseprogressing 4 months later. He is then given weekly dosing ofepratuzumab-SN-38 at an initial schedule of 6 mg/kg for 4 weeks, andthen reduced to 6 mg/kg every-other-week for an additional 6 infusions.The patient is then evaluated by blood and marrow leukemic blasts aswell as FDG studies of spleen size and bone marrow involvement, and itappears that a partial response is achieved, with concomitantimprovement in the patient's general signs and symptoms. This therapythen continues over the next 8 months, but at 5 mg/kg every other week,with 3 weeks on therapy and 2 weeks rest, and a complete remission isachieved. The patient is now being evaluated as a candidate forhematopoietic stem cell transplantation.

Example 46 Humanized RFB4-SN-38 Therapy of a Patient withRelapsed/Refractory Acute Lymphoblastic Leukemia

After failing to respond to HIDAC (high-dose ara-C therapy), this20-year-old man with precursor B-cell acute lymphoblastic leukemia isgiven humanized anti-CD22 therapy with hRFB4 IgG conjugated to SN-38(average of 6 drug molecules per IgG), at a dosing schedule of 10 mg/kgweekly for two weeks, then 1 week rest, followed by infusions of 10mg/kg every other week for an additional 5 treatments. The patient isthen evaluated for presence of blood and marrow leukemic blast cells,and shows a >90% reduction. After a rest of 4 weeks, this therapy courseis repeated, and the evaluation 4 weeks later shows a complete responsewith no minimal residual disease, as measured by PCR.

Example 47 Consolidation Therapy with Epratuzumab-SN-38 in a DLBCLPatient Receiving R-CHOP Chemotherapy

This 56-year-old woman with bilateral cervical adenopathy and cervicallymph nodes measuring 1.5 to 2.0 cm, as well as a right axillary lymphnode of 3 cm, as well as retroperitoneal and bilateral pelvic lymphnodes measuring 2.5 to 3.0 cm, is diagnosed with stage 3 diffuse largeB-cell lymphoma that is positive for CD20 and CD22. She is put on astandard R-CHOP chemotherapy regimen given every 21 days with filgrastimand prophylactic antibiotics. After receiving 6 cycles of this therapy,the patient is given a rest period of 2 months, and then is put onconsolidation therapy with 8 mg/kg epratuzumab-SN-38, infused everyother week for 3 treatments. Whereas the response after the R-CHOPchemotherapy is minimal (less than 30% change in measured lesions),consolidation therapy with epratuzumab-SN-38 results in a partialresponse (>50% decrease in sum of all index lesions). After a rest of 3months, this course of therapy with epratuzumab-SN-38 is repeated, withthe patient again given filgrastim and prophylactic antibiotics, andmaintains her good remission.

Example 48 Treatment of Relapsed Metastatic Testicular Cancer withIMMU-31-SN-38

The patient is a 30-year-old man with a history of resected testicularcancer of his right testicle, with synchronous metastases to both lungsthat respond well to combination chemotherapy. At diagnosis, his bloodtiter of alpha-fetoprotein (AFP) is elevated at 1,110 ng/mL, butdecreases to 109 ng/mL after successful therapy. He now presents with agradually rising AFP titer over a period of 3 years, so CT and FDG-PETscans of his body are made, revealing recurrence of lung metastases toboth lungs. He receives therapy with the anti-AFP antibody, IMMU-31 IgG,conjugated with SN-38 at 6 drug molecules per IgG. He receives weeklydoses of 12 mg/kg of this antibody-drug conjugate for 3 weeks of a4-week cycle, repeated for another cycle but with a reduction of thetherapeutic to 10 mg/kg. This is then repeated for another 2 cycles. Twoweeks later, radiological examination of his lungs reveals that themetastases have disappeared. His blood AFP titer is now 18 ng/mL. Thepatient returns to normal activity with a complete response having beenachieved.

Example 49 Treatment of Relapsed Metastatic Hepatocellular Carcinomawith IMMU-31-SN-38

A 58-year-old male with a history of hepatitis B infection, alcoholexcess and smoking, leads first to liver cirrhosis and then a diagnosisof hepatocellular carcinoma. At the time he presents after having aportion of his liver resected, there are also regional lymph nodesinvolved. The patient receives a course of sorafenib therapy, indicatessome general improvement, but does not have any reduction of hisregional lymph node or 2 lung (right lung) metastases. CT of the liveralso suggests that there may be a recurrence in the remaining liverparenchyma. This patient is now given 3 courses of therapy withIMMU-31-SN-38, each comprising a schedule of weekly 16 mg/kg for 2 weeksof a 4-week cycle. After the 3 courses comprising 6 doses, the patientis reevaluated and shows a decrease in his circulating AFP titer fromthe baseline value of 2,000 ng/mL to 170 ng/mL, as well as a 20%reduction of the sum of his measured index lesions. After a rest of 2months, another course of therapy of 3 cycles, but with a reduction ofthe dose to 1 mg/kg per infusion, is instituted. One month later, thereis a greater reduction of all measured lesion, to 35% of baseline, aswell as a slight decrease in the AFP blood titer to 100 ng/mL. Thepatient is going on maintenance therapy of one dose per month for aslong as there is no disease progression or limiting toxicities.

Example 50 Immunoconjugate Storage

The conjugates described in Example 8 were purified and buffer-exchangedwith 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, and furtherformulated with trehalose (25 mM final concentration) and polysorbate 80(0.01% v/v final concentration), with the final buffer concentrationbecoming 22.25 mM as a result of excipient addition. The formulatedconjugates were lyophilized and stored in sealed vials, with storage at2° C.-8° C. The lyophilized immunoconjugates were stable under thestorage conditions and maintained their physiological activities.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

We claim:
 1. A method of treating breast, lung, pancreatic, esophageal,medullary thyroid, ovarian, uterine, prostatic, testicular, colon,rectal or stomach cancer comprising administering to a human subjectwith breast, lung, pancreatic, esophageal, medullary thyroid, ovarian,uterine, prostatic, testicular, colon, rectal or stomach cancer animmunoconjugate comprising SN-38 conjugated to an hMN-14 (anti-CEACAM5)antibody or antigen-binding fragment thereof; wherein theimmunoconjugate is administered at a dosage of between 3 mg/kg and 18mg/kg.
 2. The method of claim 1, wherein the dosage is selected from thegroup consisting of 3 mg/kg, 4 mg/kg, 6 mg/kg, 8 mg/kg, 9 mg/kg, 10mg/kg, 12 mg/kg, 16 mg/kg and 18 mg/kg.
 3. The method of claim 1,wherein the immunoconjugate dosage is administered to the human subjectonce or twice a week on a schedule with a cycle selected from the groupconsisting of: (i) weekly; (ii) every other week; (iii) one week oftherapy followed by two, three or four weeks off; (iv) two weeks oftherapy followed by one, two, three or four weeks off; (v) three weeksof therapy followed by one, two, three, four or five weeks off; (vi)four weeks of therapy followed by one, two, three, four or five weeksoff; (vii) five weeks of therapy followed by one, two, three, four orfive weeks off; and (viii) monthly.
 4. The method of claim 1, whereinthe cycle is repeated 4, 6, 8, 10, 12, 16 or 20 times.
 5. The method ofclaim 1, wherein the treatment results in a reduction in tumor size ofat least 15%, at least 20%, at least 30%, or at least 40%.
 6. The methodof claim 1, wherein there is a linker between the SN-38 and theantibody.
 7. The method of claim 6, wherein the linker is CL2A and thestructure of the immunoconjugate is MAb-CL2A-SN-38


8. The method of claim 7, wherein the 10-hydroxy position of SN-38 inMAb-CL2A-SN-38 is a 10-O-ester or 10-O-carbonate derivative using a‘COR’ moiety, wherein “CO” is carbonyl and the “R” group is selectedfrom (i) an N,N-disubstituted aminoalkyl group “N(CH₃)₂—(CH₂)_(n)—”wherein n is 1-10 and wherein the terminal amino group is optionally inthe form of a quaternary salt; (ii) an alkyl residue “CH₃—(CH₂)_(n)—”wherein n is 0-10; (iii) an alkoxy moiety “CH₃—(CH₂)n-O—” wherein n is0-10; (iv) an “N(CH₃)₂—(CH₂)_(n)—O—” wherein n is 2-10; or (v) an“R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” wherein R₁ is ethyl or methyl and n isan integer with values of 0-10.
 9. The method of claim 1, wherein theimmunoconjugate is administered in combination with one or moretherapeutic modalities selected from the group consisting ofunconjugated antibodies, radiolabeled antibodies, drug-conjugatedantibodies, toxin-conjugated antibodies, gene therapy, chemotherapy,therapeutic peptides, cytokine therapy, oligonucleotides, localizedradiation therapy, surgery and interference RNA therapy.
 10. The methodof claim 9, wherein the drug, toxin or chemotherapeutic agent isselected from the group consisting of 5-fluorouracil, afatinib, aplidin,azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291,bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan,calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin,carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors,irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans,cyclophosphamide, crizotinib, cytarabine, dacarbazine, dasatinib,dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptorbinding agents, etoposide (VP16), etoposide glucuronide, etoposidephosphate, exemestane, fingolimod, flavopiridol, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, fostamatinib, ganetespib,GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib,idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib,lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin,SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC),transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839.
 11. The method of claim 1,wherein the immunoconjugate is stored at pH 6 to 7 in a pharmaceuticalcomposition comprising a buffer selected from the group consisting ofN-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (MES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes].
 12. The method ofclaim 11, wherein the composition further comprises 25 mM trehalose and0.01% v/v polysorbate
 80. 13. The method of claim 1, wherein theimmunoconjugate comprises 4 or more molecules of SN-38 conjugated to theantibody or antigen-binding fragment thereof.
 14. The method of claim 1,wherein there are 6 or more SN-38 molecules attached to each antibodymolecule.
 15. The method of claim 1, wherein there are 1 to 5 SN-38molecules attached to each antibody molecule.
 16. The method of claim 1,wherein the cancer is metastatic.
 17. The method of claim 16, furthercomprising reducing in size or eliminating the metastases.
 18. Themethod of claim 1, wherein the cancer is refractory to other therapiesbut responds to the immunoconjugate.
 19. The method of claim 1, whereinthe patient has failed to respond to at least one other therapy, priorto treatment with the immunoconjugate.
 20. The method of claim 19,wherein the patient has failed to respond to therapy with irinotecan,prior to treatment with the immunoconjugate.
 21. The method of claim 20,wherein the cancer is metastatic colon cancer and the patient has failedtherapy with irinotecan, oxaliplatin, FOLFIRINOX, FOLFIRI, FOLFOX,bevacizumab, cetuximab, ramucirumab, 5-fluorouracil or leucovorin priorto administration of the immunoconjugate.
 22. The method of claim 1,further comprising administering to the subject an anti-CEACAM6-SN-38immunoconjugate.